Compositions and method of treating cancer

ABSTRACT

The disclosure provides for methods and compositions useful for treating infections, cancer and neoplastic diseases and disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 from ProvisionalApplication Ser. No. 62/477,370, filed Mar. 27, 2017, and fromProvisional Application Ser. No. 62/638,058, filed Mar. 2, 2018, thedisclosures of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No.AI083358, awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

TECHNICAL FIELD

The disclosure relates to anti-cancer agents, methods of making, andmethods of use thereof.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

Accompanying this filing is a Sequence Listing entitled “SequenceST25.txt”, created on Mar. 26, 2018 and having 98,239 bytes of data,machine formatted on IBM-PC, MS-Windows operating system. The sequencelisting is hereby incorporated herein by reference in its entirety forall purposes.

Microrganism Deposit

Exemplary microorganisms of the disclosure (Staphylococcus epidermidisM034 and Staphylococcus epidermidis M038) were deposited on Mar. 22,2018 with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, as ATCC Number ______ (straindesignation S. epi-M038 UCSD 20180315) and as ATCC Number ______ (straindesignation S. epi-M034 UCSD 20180315) under the Budapest Treaty. Thisdeposit will be maintained at an authorized depository and replaced inthe event of mutation, nonviability or destruction for a period of atleast five years after the most recent request for release of a samplewas received by the depository, for a period of at least thirty yearsafter the date of the deposit, or during the enforceable life of therelated patent, whichever period is longest. All restrictions on theavailability to the public of these cell lines will be irrevocablyremoved upon the issuance of a patent from the application.

BACKGROUND

It is estimated that in 2017 there will be over 1.5 mllion new cancercases diagnosed and greater than 600,000 cancer deaths in the U.S.Although advances have been made to treat and prevent various cancers,new methods and compositions are need.

SUMMARY

The disclosure provides compositions and methods useful for thetreatment of neoplasias and cancers.

In a particular embodiment, the disclosure provides for a compoundhaving the general formula of Formula I(a):

or a pharmaceutically acceptable salt or prodrug thereof, wherein, N¹-N⁵are nitrogen atoms; X¹-X² are carbon atoms; the R groups attached by adashed line are present, or are not present if the R group is connectedto an atom that is bound to another atom by a double covalent bond; thebond indicated by both a straight line and a dashed line indicate thatthe bond may be a single covalent bond or a double covalent bond; thefused heterocyclic ring system comprises three double bonds with N² orN³ forming a double bond and with X¹, and with N⁴ or N⁵ forming a doublebond with X²; R¹ is a hydroxyl, ester, carboxylic acid, or —O—R¹⁰; R²,R⁴, R⁵, R⁷-R⁹ are independently a H, D, optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-alkenyl, optionallysubstituted (C₁-C₆)-alkynyl, optionally substituted (C₃-C₁₂)cycloalkyl,optionally substituted (C₄-C₁₂)cycloalkenyl, optionally substitutedaryl; R³ and R⁶ are independently selected from a H, D, optionallysubstituted (C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-heteroalkyl,optionally substituted (C₁-C₆)-alkenyl, optionally substituted(C₁-C₆)-heteroalkenyl, optionally substituted (C₁-C₆)-alkynyl,optionally substituted (C₁-C₆)-heteroalkynyl, optionally substituted(C₃-C₁₂)cycloalkyl, optionally substituted (C₄-C₁₂)cycloalkenyl,optionally substituted aryl, optionally substituted heterocycle, halide,hydroxyl, carbonyl, aldehyde, carboxyl, ester, alkoxy, carboxyamide,amine, imine, azide, cyano, nitro, nitroso, thiol, sulfide, sulfoxide,sulfone, and phosphate; R¹⁰ is selected from D, optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-heteroalkyl, optionallysubstituted (C₁-C₆)-alkenyl, optionally substituted(C₁-C₆)-heteroalkenyl, optionally substituted (C₁-C₆)-alkynyl,optionally substituted (C₁-C₆)-heteroalkynyl, optionally substituted(C₃-C₁₂)cycloalkyl, optionally substituted (C₄-C₁₂)cycloalkenyl,optionally substituted aryl, and optionally substituted heterocycle.

In another embodiment, the disclosure provides a compound having thegeneral formula of Formula I(b):

or a pharmaceutically acceptable salt or prodrug thereof, wherein, N¹-N⁵are nitrogen atoms; X¹—X² are carbon atoms; the R groups attached by adashed line are present, or are not present if the R group is connectedto an atom that is bound to another atom by a double covalent bond; thebond indicated by both a straight line and a dashed line indicate thatthe bond may be a single covalent bond or a double covalent bond; thefused heterocyclic ring system comprises three double bonds with N² orN³ forming a double bond with X¹, and with N⁴ or N⁵ forming a doublebond with X²; R¹ is a hydroxyl, ester, carboxylic acid, or —O—R¹⁰; R²,R⁴, R⁵, and R⁷ are independently a H, D, optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-alkenyl, optionallysubstituted (C₁-C₆)-alkynyl, optionally substituted (C₃-C₁₂)cycloalkyl,optionally substituted (C₄-C₁₂)cycloalkenyl, optionally substitutedaryl; R¹⁰ is selected from D, optionally substituted (C₁-C₆)-alkyl,optionally substituted (C₁-C₆)-heteroalkyl, optionally substituted(C₁-C₆)-alkenyl, optionally substituted (C₁-C₆)-heteroalkenyl,optionally substituted (C₁-C₆)-alkynyl, optionally substituted(C₁-C₆)-heteroalkynyl, optionally substituted (C₃-C₁₂)cycloalkyl,optionally substituted (C₄-C₁₂)cycloalkenyl, optionally substitutedaryl, and optionally substituted heterocycle.

In yet another embodiment, the disclosure provides a compound of generalformula II (6-N-hydroxyaminopurine (6-HAP)):

or a pharmaceutically acceptable salt or prodrug thereof; or a tautomerof the compound of Formula II, or a pharmaceutically acceptable salt orprodrug of the tautomer of compound of Formula II thereof.

In another embodiment, the disclosure provides a pharmaceuticalcomposition comprising a compound of formula I(a), I(b) and/or II and apharmaceutically acceptable carrier. In yet another embodiment, thepharmaceutical composition comprises at least one additional activeagent. In still a further embodiment, the at least one additional activeagent is a chemotherapeutic agent. If still a further embodiment, thechemotherapeutic agent is selected from the group consisting of analkalating agent, an antimetabolite, an anti-microtubule agent, atopoisomerase inhibitor, and a cytotoxic antibiotic. In a furtherembodiment, the anticancer agent is selected from the group consistingof cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen,raloxifene, estrogen receptor binding agents, docetaxel, paclitaxel,gemcitabine, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristine, vinblastine,6-mercaptopurine; capecitabine; cladribine; clorfarabine; cytarabine;doxorubicin; fludarabine; floxuridine; gemcitabine; hydroxyurea;methotrexate; pemetrexed; pentostatin; prednisone; procarbazineandmethotrexate, or any analog or derivative variant thereof.

The disclosure also provides a method of treating a neoplasm (includingprecancerous), cell proliferative disorder or cancer comprisingcontacting a subject topically or parenterally with a compound orpharmaceutical composition containing the compound of Formula I(a), I(b)and/or II in an amount effective to treat the neoplasm, cancer or cellproliferative disorder.

S. epidermidis produces 6-HAP, which was found to inhibit DNA synthesisand has the potential to convey protection against neoplasia. Abeneficial role for skin bacteria in host defense is consistent withobservations of a role for commensal bacteria to resist S. aureusinfection, but further extends this concept to host defense functionsagainst cancer and pre-cancerous neoplasms, e.g., papillomas and actinickeratosis). Moreover, a loss of S. epidermidis strains that produce6-HAP may increase a subject's risk for developing skin cancer. As suchthe disclosure provides for probiotic compositions which comprise an S.epidermidis strains that produces 6-HAP for preventing, attenuatingand/or inhibiting neoplasia in a subject.

In a particular embodiment, the disclosure provides for a compositionthat protects a subject from skin cancer or other type of neoplasia,comprising (i) a composition of formulat I, II or II and/or (ii) aprobiotic commensal microorganism that produces 6-N-hydroxyaminopurine(6-HAP). In a further embodiment, the probiotic commensal microorganismis a strain of Staphylococcus epidermis. In yet a further embodiment,the strain is one or more of Staphylococcus epidermis is Staphylococcusepidermis M034 and/or Staphylococcus epidermis M038. In anotherembodiment, the composition is formulated for topical or dermaldelivery. In yet another embodiment, the composition is in the form of alotion, shake lotion, cream, ointment, gel, foam, powder, solid, pasteor tincture. In a further embodiment, the composition further comprisesone or more sunscreen agents. Examples of sunscreen agents include, butare not limited to, aminobenzoic acid, avobenzone, cinoxate,dioxybenzone, ecamsule, ensulizole, homosalate, meradimate, octocrylene,octinoxate, octisalate, oxybenzone, padimate 0, sulisobenzone, titaniumdioxide, trolamine salicylate, and zinc oxide. In yet a furtherembodiment, the skin protectant composition further comprises one ormore topical antibiotics. Examples of topical antiboitics include, butare not limited to, sulfacetamide sodium, bacitracin, polymyxin b,erythromycin, silver sulfadiazine, neomycin, retapamulin, and mupirocin.

In a certain embodiment, the disclosure further provides for a method ofpreventing a subject from developing skin cancer or other type ofneoplasia, comprising topically administering to the subject acomposition of the disclosure. In a further embodiment, the neoplasia isan epithelial neoplasia. In yet a further embodiment, the neoplasia isinduced by or from UV exposure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D shows S. epidermidis strains isolated from normal human skinproduce non-proteinaceous molecule with antimicrobial activity. (a)Screening for antimicrobial activity of culture supernatant from 44strains of S. epidermidis strains isolated from normal human skin byradial diffusion assay against gropu A streptococcus (GAS). Datarepresent diameter of growth inhibition zone of conditioned media ofeach strain. UD=undetectable. (b) Elution profile of the antimicrobialcompound purified from culture supernatant of S. epidermidis M034 byHPLC using a PolyHYDROXYMETHYL. The last step of 5 purification steps isshown. The insert panel represents antimicrobial activity of eachfraction on radial diffusion assay against GAS. Green line represents agradient of H₂O in acetonitrile. (c-d) Stability of antimicrobialmolecules from S. epidermidis producing the strongest antibioticactivity (M034) against GAS after heat-treatment (100° C. for indicatedtime) (c) and incubation with proteinase K (2 mg/mL) or papain (2 mg/mL)at 37° C. for 3 hrs, followed by 5-min incubation at 90° C. toinactivate enzyme (d). Antimicrobial activity against GAS was determinedby radial diffusion assay.

FIG. 2A-B shows colonization by S. epidermidis strain producing 6-HAPenhances antimicrobial activity against pathogenic bacteria on skinsurface. (a-b) Antimicrobial activity of mouse skin colonized by eitherS. epidermidis producing 6-HAP (M034), a non-antimicrobial strain(ATCC1457), or vehicle against GAS or methicillin resistantStaphylococcus aureus (MRSA) challenge. Mouse dorsal skin was appliedwith S. epidermidis (M034 or ATCC1457) or PBS (vehicle) for 2 hrs.Pathogens were then applied to the skin surface for 6 hrs. Bacterialsurvival was measured by swabbing and counting serial dilutions of theswab sample plated on a blood agar plate for GAS (hemolytic) and S.epidermidis (non-hemolytic) or a mannitol salt agar with egg yolk forMRSA (mannitol positive: a large yellow colony with egg yolk reaction)and S. epidermidis (mannitol negative: a small pink colony without eggyolk reaction). Each dot represents data from an individual mouse.*P<0.05 and **P<0.01 by Student's t-test.

FIG. 3A-G shows S. epidermidis strains produce 6-N-hydroxyaminopurinewith antimicrobial activity. (a) Molecular mass of purified antibioticfrom S. epidermidis M034 strain analyzed by high-resolution electronspray ionization mass spectrometry. (b) ¹⁵N isotope incorporation intothe antibiotic molecule after culturing S. epidermidis M034 in TSBcontaining ammonium-¹⁵N chloride (12.5 mM) for 24 hrs. (c-d) Comparisonof chemical shifts of purified antibiotic (c) with those of synthetic6-HAP (d) in ¹H-NMR. (e-f) Comparison of the fragmentation profile ofpurified antibiotic (e) with that of synthetic 6-HAP (f) onelectron-impact mass spectrometry. (g) The determined chemical structureof the antibiotic (6-HAP).

FIG. 4A-G shows 6-HAP is a direct inhibitor of DNA polymerization. (a)Time course killing of GAS in THB containing 6-HAP (25 μg/mL), mitomycinC (5 μg/mL), or LL-37 (10 μM). At each indicated time point, live GAS inthe media was measured by counting CFU on an agar plate. The datarepresent mean±SE of four individual experiments. (b) Membranepermeability of GAS incubated in THB containing 6-HAP (25 μg/mL),mitomycin C (5 μg/mL) and LL-37 (10 μM) for 1 hr. The bacteria withcompromised plasma membranes (Red) can be distinguished from those withintact membranes (Green). (c) BrdU incorporation into nascent DNA of GASafter incubation with 6-HAP (25 μg/mL) or mitomycin C (5 μg/mL) in THBcontaining BrdU (10 μM) for 30 or 60 min. (d) BrdU incorporation intonascent DNA of S. epidermidis ATCC12228 after incubation with 6-HAP (25μg/mL) or mitomycin C (5 μg/mL) in THB containing BrdU (10 μM) for 60min. The data represent mean±SE of five individual experiments (*P<0.05and **P<0.01 by Student's t-test vs vehicle control). (e-f) Capacity of6-HAP to block in vitro DNA extension by Klenow fragment polymerase.Extension reaction was carried out with IRDye800-labeled 18-nt primer(SEQ ID NO:57) and 25-nt template which required adenosine (X=T) orcytidine (X=G) at the initial base for extension (SEQ ID NO:58) (e).Extension reaction was analyzed on a 20% acrylamide gel byelectrophoresis (f). (g) Antimicrobial activity of 6-HAP against GAS inthe presence of adenine. GAS was incubated in media containing 6-HAPwith or without adenine for 20 hrs. GAS survival was measured bycounting CFU. The data represent mean±SE of four individual experiments(**P<0.01 by Student's t-test).

FIG. 5A-E shows mARC2 responsible for selective anti-proliferativeactivity of 6-HAP. (a-b) Selective antiproliferative activity of 6-HAPagainst Pam212 squamous cell carcinoma, but not against normalkeratinocytes. BrdU incorporation into Pam212 cells (b) or NHEKs (b)after 4-hr or 24-hr incubation, respectively, in suitable mediacontaining indicated concentrations of 6-HAP or mitomycin C (10 μg/mL)(a). (c) Expression of mARC1 and mARC2 in NHEKs, squamous cell carcinoma(Pam212), melanoma (B16F10) and lymphoma cell lines (L5178). To comparerelative expression level in each cells, data was shown as relative toGAPDH expression. (d) Expression of mARC1 and mARC2 in NHEKs treatedwith control siRNA, mARC1 siRNA and mARC2 siRNA. (e) Effect of genesilencing with mARC1 and mARC2 siRNA on sensitivity to 6-HAP in NHEKs.NHEKs treated with each siRNA were incubated for 48 hrs and thenincubated with 10 ng/mL of 6-HAP for 24 hrs.

FIG. 6A-E shows 6-HAP improves deep skin infection and slows growth ofB16 melanoma in mice. (a-c) Effect of a single intravascular injectionof 6-HAP on skin infection by GAS. The size of the infected lesion wasmeasured by Image-J software (a). Representative images of infected skin(arrow) of mouse treated with 6-HAP or vehicle at Day-1 and Day-3 postinfection are shown in (b). Infected skin was removed 24 and 72 hrsafter bacterial injection and homogenized in PBS (c). CFUs wereenumerated by plating serial dilutions of the homogenate on an agarplate. The data represent mean±SE of eight individual experiments(*P<0.05 and **P<0.01 by Student's t-test vs vehicle control). (d-e)Effect of repeated intravascular administrations with 6-HAP on growth ofmelanoma in mice. The data represent mean±SE of 10 individualexperiments (*P<0.05, **P<0.01 and ***P<0.001 by Student's t-test vsvehicle control) (d). Representative images of tumor (broken line) inmouse treated with 6-HAP or vehicle at Day-9 and Day-13 are shown in(e).

FIG. 7A-H shows S. epidermidis strains producing 6-N-hydroxyaminopurinesuppress UV-induced skin tumor formation in SKH-1 hairless mice. (a-d)Effect of colonization by S. epidermidis M034 producing 6-HAP on tumorincidence (a) and number (b) in SKH-1 hairless mice treated with DMBA,followed by UV-B irradiation at 180 mJ/cm² twice a week. S. epidermidisATCC1457 was used as a control strain that does not produce 6-HAP. Tumorincidence and tumor number in each mouse were recoded every week. Thedata represent mean±SE of 19 mice. Representative images of UV-inducedtumor formation in mouse treated with S. epidermidis ATCC1457 (c) orM034 (d) at week-12 are shown. (e-f) A representative H&E staining ofUV-induced skin tumor or skin obtained from SKH-1 mice colonized by S.epidermidis 1457 (e) or M034 (f), respectively, treated with UV-B for 12weeks. (g-h) Immunostaining for S. epidermidis and keratin-14 in theUV-induced tumor or skin of SKH-1 mice treated with S. epidermidisATCC1457 (g) or M034 (h), respectively.

FIG. 8A-D shows Productions of 6-HAP by skin isolate strains andlaboratory strains of S. epidermidis. (a-d) M034 (a) and M038 (b)strains of S. epidermidis isolated from the surface of normal humanskin, or ATCC12228 (c) and ATCC1457 (d) laboratory strains were culturedovernight in TSB. 6-HAP was partially purified from culture supernatantaccording to the Method section. Left panel shows a HPLC-elution profileof 6-HAP (Arrow) on a TSKgel NH2-100 amino column (4.6×150 mm) (TosohBiosci. LLC, Tokyo, Japan) after Sep-Pak step (See method). The elutionprofile was monitored at 270 nm. Green line represents a gradient of H₂Oin acetonitrile. The right panel represents antimicrobial activity ofeach fraction on radial diffusion assay against GAS.

FIG. 9 shows the gHMBC Spectrum (500 MHz) of 6-HAP in AcOD-D2O. Thecarbon spectrum of 6-HAP was measured indirectly by the gHMBCexperiment. The gHMBC spectral data was recorded on a Mercury Pluss 500(Varian) spectrometer. FID file was processed using MestRenova 8.1(MestreLab Research). The gHMBC spectrum of 6-HAP in AcOD-D2O (1:5 v/v)revealed five carbon signals in the aromatic region (δC=113.60, 144.94,148.17, 150.28, 150.45).

FIG. 10 shows a comparison of antimicrobial activity of natural 6-HAPand synthetic 6-HAP. GAS (1×10⁵ CFU/mL) were incubated with indicatedconcentrations of purified 6-HAP or synthetic 6-HAP in THB overnight.Bacterial growth was monitored by measuring OD600 (relative % of growthindex).

FIG. 11 shows the capacity of 6-HAP to directly disrupt plasma membraneof human keratinocytes and sebocytes. Normal human epidermalkeratinocytes (NHEKs) or immortalized human sebocyte cell line (SZ95)(1×10⁵ cells) were incubated with the indicated concentrations of 6-HAPin Epilife or Sebmed medium, respectively, at 37° C. for 6 hrs. Vehicle(0.5% DMSO) or Triton X-100 (0.1%) was added to achieve 0% or 100% ofLDH release, respectively. LDH release was determined with CytotoxicityDetection Kit (LDH) (Roche, Mannheim, Germany) according to the protocolprovided. Data represent mean±SE of three individual experiments.

FIG. 12A-C shows 6-HAP exerts antiproliferative activity against tumorcell lines. Proliferative activity of tumor cell line, L5178 (a), YAC-1lymphoma (b), B16F10 melanoma (c) after 4-hr incubation in suitablemedia containing indicated concentrations of 6-HAP or mitomycin C (10μg/mL). Proliferative activity of cells was determined by monitoringBrdU incorporation. The data represent mean±SE of four individualexperiments.

FIG. 13 shows Systemic toxicity of 6-HAP in mice. C57BL6 mice (8 weekfemale) were intravascularly administrated with 6-HAP (20 mg/kg) or withan equal volume of vehicle (2.5% DMSO in 0.9% NaCl) every 48 hours for 2weeks (Arrows). To observe toxicity of 6-HAP, mouse weight wasdetermined at indicated time points. Data represent mean±SE of 10 mice.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a compound” includes aplurality of such compounds and reference to “the cell” includesreference to one or more cells and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods andreagents similar or equivalent to those described herein can be used inthe practice of the disclosed methods and compositions, the exemplarymethods and materials are now described.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,”“including,” “have,” “haves,” and “having” are interchangeable and notintended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the methodologies,which are described in the publications, which might be used inconnection with the description herein. However, with respect to anysimilar or identical terms found in both the incorporated publicationsor references and those expressly put forth or defined in thisapplication, then those terms definitions or meanings expressly putforth in this application shall control in all respects. Thepublications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The term “alkyl”, refers to an organic group that is comprised of carbonand hydrogen atoms that contains single covalent bonds between thecarbons. Generally, an “alkyl” as used in this disclosure, refers to anorganic group that contains 1 to 20 carbon atoms, unless statedotherwise. Wherein if there is more than 1 carbon, the carbons may beconnected in a linear manner, or alternatively if there are more than 2carbons then the carbons may also be linked in a branched fashion sothat the parent chain contains one or more secondary, tertiary, orquaternary carbons. An alkyl may be substituted or unsubstituted, unlessstated otherwise. Substituted alkyl groups include among others thosewhich are substituted with aryl groups, which in turn can be optionallysubstituted. Specific alkyl groups include methyl, ethyl, n-propyl,iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl,n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, andcyclohexyl groups, all of which are optionally substituted. Specificsubstituted alkyl groups include haloalkyl groups, particularlytrihalomethyl groups and specifically trifluoromethyl groups.

The term “alkenyl”, refers to an organic group that is comprised ofcarbon and hydrogen atoms that contains at least one double covalentbond between two carbons. Generally, an “alkenyl” as used in thisdisclosure, refers to organic group that contains 1 to 20 carbon atoms,unless stated otherwise. While a C₁-alkenyl can form a double bond to anatom of a parent chain, an alkenyl group of three or more carbons cancontain more than one double bond. It certain instances the alkenylgroup will be conjugated, in other cases an alkenyl group will not beconjugated, and yet other cases the alkenyl group may have stretches ofconjugation and stretches of nonconjugation. Additionally, if there ismore than 1 carbon, the carbons may be connected in a linear manner, oralternatively if there are more than 3 carbons then the carbons may alsobe linked in a branched fashion so that the parent chain contains one ormore secondary, tertiary, or quaternary carbons. An alkenyl may besubstituted or unsubstituted, unless stated otherwise. Substitutedalkenyl groups include among others those which are substituted withalkyl or aryl groups, which groups in turn can be optionallysubstituted. Specific alkenyl groups include ethenyl, prop-1-enyl,prop-2-enyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, branchedpentenyl, hex-1-enyl, branched hexenyl, all of which are optionallysubstituted.

The term “alkynyl”, refers to an organic group that is comprised ofcarbon and hydrogen atoms that contains a triple covalent bond betweentwo carbons. Generally, an “alkynyl” as used in this disclosure, refersto organic group that contains 1 to 20 carbon atoms, unless statedotherwise. While a C₁-alkynyl can form a triple bond to an atom of aparent chain, an alkynyl group of three or more carbons can contain morethan one triple bond. Where if there is more than 1 carbon, the carbonsmay be connected in a linear manner, or alternatively if there are morethan 4 carbons then the carbons may also be linked in a branched fashionso that the parent chain contains one or more secondary, tertiary, orquaternary carbons. An alkynyl may be substituted or unsubstituted,unless stated otherwise.

The term “antimicrobial” as it relates to treatments, agents, andcompounds refers to an agent that can be used to suppress, attenuate,ameliorate, any symptom caused by or resulting from an infection by aforeign agent. For the purposes of this disclosure a foreign agentincludes, but is not limited to, bacteria, parasites, viruses, andfungi.

The term “anticancer” as it relates to treatments, agents, and compoundsrefers to an agent (e.g., small molecule such as 6-HAP or a probiotic)that can be used to suppress, attenuate, ameliorate, any symptom causedby or resulting from a cell proliferative disorder, neoplasm or cancer.

The term “aryl”, as used in this disclosure, refers to a conjugatedplanar ring system with delocalized pi electron clouds that contain onlycarbon as ring atoms. An “aryl” for the purposes of this disclosureencompass from 1 to 7 aryl rings wherein when the aryl is greater than 1ring the aryl rings are joined so that they are linked, fused, or acombination thereof. An aryl may be substituted or unsubstituted, or inthe case of more than one aryl ring, one or more rings may beunsubstituted, one or more rings may be substituted, or a combinationthereof. Substituted aryl groups include among others those which aresubstituted with alkyl or alkenyl groups, which groups in turn can beoptionally substituted. Specific substituted aryl groups include mono-,di-, tri, tetra- and pentahalo-substituted phenyl groups; mono-, di-,tri-, tetra-, penta-, hexa-, and hepta-halo-substituted naphthalenegroups; 3- or 4-halo-substituted phenyl groups, 3- or4-alkyl-substituted phenyl groups, 3- or 4-alkoxy-substituted phenylgroups, 3- or 4-RCO-substituted phenyl, 5- or 6-halo-substitutednaphthalene groups. More specifically, substituted aryl groups includeacetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenylgroups, particularly 3-fluorophenyl and 4-fluorophenyl groups;chlorophenyl groups, particularly 3-chlorophenyl and 4-chlorophenylgroups; methylphenyl groups, particularly 4-methylphenyl groups, andmethoxyphenyl groups, particularly 4-methoxyphenyl groups. Specific arylgroups include phenyl groups, biphenyl groups, and naphthyl groups, allof which are optionally substituted.

For purposes of the disclosure the term “cancer” will be used toencompass cell proliferative disorders, neoplasms, precancerous celldisorders and cancers. Thus, a “cancer” refers to any cell thatundergoes aberrant cell proliferation that can lead to metastasis ortumor growth. Exemplary cancers include but are not limited to,adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma,anal cancer, anorectal cancer, cancer of the anal canal, appendixcancer, childhood cerebellar astrocytoma, childhood cerebralastrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliarycancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer,bladder cancer, urinary bladder cancer, bone and joint cancer,osteosarcoma and malignant fibrous histiocytoma, brain cancer, braintumor, brain stem glioma, cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodeimal tumors, visual pathway andhypothalamic glioma, breast cancer, including triple negative breastcancer, bronchial adenomas/carcinoids, carcinoid tumor,gastrointestinal, nervous system cancer, nervous system lymphoma,central nervous system cancer, central nervous system lymphoma, cervicalcancer, childhood cancers, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic myeloproliferative disorders, coloncancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm,mycosis fungoides, Seziary Syndrome, endometrial cancer, esophagealcancer, extracranial germ cell tumor, extragonadal germ cell tumor,extrahepatic bile duct cancer, eye cancer, intraocular melanoma,retinoblastoma, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),germ cell tumor, ovarian germ cell tumor, gestational trophoblastictumor glioma, head and neck cancer, hepatocellular (liver) cancer,Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocularcancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidneycancer, renal cancer, laryngeal cancer, acute lymphoblastic leukemia,acute myeloid leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer,liver cancer, lung cancer, non-small cell lung cancer, small cell lungcancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary centralnervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma,melanoma, intraocular (eye) melanoma, merkel cell carcinoma,mesothelioma malignant, mesothelioma, metastatic squamous neck cancer,mouth cancer, cancer of the tongue, multiple endocrine neoplasiasyndrome, mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, chronic my elogenousleukemia, acute myeloid leukemia, multiple myeloma, chronicmyeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oralcancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer,ovarian epithelial cancer, ovarian low malignant potential tumor,pancreatic cancer, islet cell pancreatic cancer, paranasal sinus andnasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiplemyeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renalpelvis and ureter, transitional cell cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, ewing family of sarcoma tumors,soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer(non-melanoma), skin cancer (melanoma), papillomas, actinic keratosisand keratoacanthomas, merkel cell skin carcinoma, small intestinecancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric)cancer, supratentorial primitive neuroectodermal tumors, testicularcancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroidcancer, transitional cell cancer of the renal pelvis and ureter andother urinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, and Wilm's Tumor. In some embodiments,the cancer is selected from the group consisting of melanoma, colorectalcancer, pancreatic cancer, bladder cancer, breast cancer, triplenegative breast cancer, ovarian cancer and lung cancer.

The term “cylcloalkyl”, as used in this disclosure, refers to an alkylthat contains at least 3 carbon atoms but no more than 12 carbon atomsconnected so that it forms a ring. A “cycloalkyl” for the purposes ofthis disclosure encompass from 1 to 7 cycloalkyl rings, wherein when thecycloalkyl is greater than 1 ring, then the cycloalkyl rings are joinedso that they are linked, fused, or a combination thereof. A “cycloalkyl”can also include bicyclic and tricyclic-based groups. A cycloalkyl maybe substituted or unsubstituted, or in the case of more than onecycloalkyl ring, one or more rings may be unsubstituted, one or morerings may be substituted, or a combination thereof.

The term “cylcloalkenyl”, as used in this disclosure, refers to analkene that contains at least 3 carbon atoms but no more than 12 carbonatoms connected so that it forms a ring. A “cycloalkenyl” for thepurposes of this disclosure encompass from 1 to 7 cycloalkenyl rings,wherein when the cycloalkenyl is greater than 1 ring, then thecycloalkenyl rings are joined so that they are linked, fused, or acombination thereof “Cycloalkenyl” can include bicyclic andtricyclic-based groups. A cycloalkenyl may be substituted orunsubstituted, or in the case of more than one cycloalkenyl ring, one ormore rings may be unsubstituted, one or more rings may be substituted,or a combination thereof. Specific alkenyl groups includecycloprop-1-enyl, cyclobut-1-enyl, cyclobut-2-enyl, cyclopent-1-enyl,cyclohexenyl, all of which are optionally substituted.

The term “heterocycle”, as used in this disclosure, refers to ringstructures that contain at least 1 noncarbon ring atom. A “heterocycle”for the purposes of this disclosure encompass from 1 to 7 heterocyclerings wherein when the heterocycle is greater than 1 ring theheterocycle rings are joined so that they are linked, fused, or acombination thereof. A heterocycle may be a hetero-aryl or nonaromatic,or in the case of more than one heterocycle ring, one or more rings maybe nonaromatic, one or more rings may be hetero-aryls, or a combinationthereof. A heterocycle may be substituted or unsubstituted, or in thecase of more than one heterocycle ring one or more rings may beunsubstituted, one or more rings may be substituted, or a combinationthereof. Typically, the noncarbon ring atom is N, O, S, Si, Al, B, or P.In case where there is more than one noncarbon ring atom, thesenoncarbon ring atoms can either be the same element, or combination ofdifferent elements, such as N and O. Examples of heterocycles include,but are not limited to: a monocyclic heterocycle such as, aziridine,oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline,imidazolidine, pyrazolidine, pyrazoline, dioxolane, sulfolane2,3-dihydrofuran, 2,5-dihydrofuran tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydro-pyridine, piperazine, morpholine,thiomorpholine, pyran, thiopyran, 2,3-dihydropyran, tetrahydropyran,1,4-dihydropyridine, 1,4-dioxane, 1,3-dioxane, dioxane, homopiperidine,2,3,4,7-tetrahydro-1H-azepine homopiperazine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethylene oxide; and polycyclicheterocycles such as, indole, indoline, isoindoline, quinoline,tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline,1,4-benzodioxan, coumarin, dihydrocoumarin, benzofuran,2,3-dihydrobenzofuran, isobenzofuran, chromene, chroman, isochroman,xanthene, phenoxathiin, thianthrene, indolizine, isoindole, indazole,purine, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, phenanthridine, perimidine, phenanthroline, phenazine,phenothiazine, phenoxazine, 1,2-benzisoxazole, benzothiophene,benzoxazole, benzthiazole, benzimidazole, benztriazole, thioxanthine,carbazole, carboline, acridine, pyrolizidine, and quinolizidine. Inaddition to the polycyclic heterocycles described above, heterocycleincludes polycyclic heterocycles wherein the ring fusion between two ormore rings includes more than one bond common to both rings and morethan two atoms common to both rings. Examples of such bridgedheterocycles include quinuclidine, diazabicyclo[2.2.1]heptane and7-oxabicyclo[2.2.1]heptane.

The terms “heterocyclic group”, “heterocyclic moiety”, “heterocyclic”,or “heterocyclo” used alone or as a suffix or prefix, refers to aheterocycle that has had one or more hydrogens removed therefrom.

The term “heterocyclyl” used alone or as a suffix or prefix, refers amonovalent radical derived from a heterocycle by removing a hydrogentherefrom. Heterocyclyl includes, for example, monocyclic heterocyclyls,such as, aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl,thietanyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, pyrazolidinyl,pyrazolinyl, dioxolanyl, sulfolanyl, 2,3-dihydrofuranyl,2,5-dihydrofuranyl, tetrahydrofuranyl, thiophanyl, piperidinyl,1,2,3,6-tetrahydro-pyridinyl, piperazinyl, morpholinyl, thiomorpholinyl,pyranyl, thiopyranyl, 2,3-dihydropyranyl, tetrahydropyranyl,1,4-dihydropyridinyl, 1,4-dioxanyl, 1,3-dioxanyl, dioxanyl,homopiperidinyl, 2,3,4,7-tetrahydro-1H-azepinyl, homopiperazinyl,1,3-dioxepanyl, 4,7-dihydro-1,3-dioxepinyl, and hexamethylene oxidyl. Inaddition, heterocyclyl includes aromatic heterocyclyls or heteroaryl,for example, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl,furyl, furazanyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl,isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl,1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl,1,3,4-thiadiazolyl, and 1,3,4 oxadiazolyl. Additionally, heterocyclylencompasses polycyclic heterocyclyls (including both aromatic ornon-aromatic), for example, indolyl, indolinyl, isoindolinyl,quinolinyl, tetrahydroquinolinyl, isoquinolinyl,tetrahydroisoquinolinyl, 1,4-benzodioxanyl, coumarinyl,dihydrocoumarinyl, benzofuranyl, 2,3-dihydrobenzofuranyl,isobenzofuranyl, chromenyl, chromanyl, isochromanyl, xanthenyl,phenoxathiinyl, thianthrenyl, indolizinyl, isoindolyl, indazolyl,purinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,cinnolinyl, pteridinyl, phenanthridinyl, perimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxazinyl, 1,2-benzisoxazolyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benzimidazolyl,benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl,pyrolizidinyl, and quinolizidinyl. In addition to the polycyclicheterocyclyls described above, heterocyclyl includes polycyclicheterocyclyls wherein the ring fusion between two or more rings includesmore than one bond common to both rings and more than two atoms commonto both rings. Examples of such bridged heterocycles include, but arenot limited to, quinuclidinyl, diazabicyclo[2.2.1]heptyl; and7-oxabicyclo[2.2.1]heptyl.

The term “hetero-aryl” used alone or as a suffix or prefix, refers to aheterocycle or heterocyclyl having aromatic character. Examples ofheteroaryls include, but are not limited to, pyridine, pyrazine,pyrimidine, pyridazine, thiophene, furan, furazan, pyrrole, imidazole,thiazole, oxazole, pyrazole, isothiazole, isoxazole, 1,2,3-triazole,tetrazole, 1,2,3-thiadiazole, 1,2,3-oxadiazole, 1,2,4-triazole,1,2,4-thiadiazole, 1,2,4-oxadiazole, 1,3,4-triazole, 1,3,4-thiadiazole,and 1,3,4-oxadiazole.

The term “hetero-” when used as a prefix, such as, hetero-alkyl,hetero-alkenyl, hetero-alkynyl, or hetero-hydrocarbon, for the purposeof this disclosure refers to the specified hydrocarbon having one ormore carbon atoms replaced by non-carbon atoms as part of the parentchain. Examples of such non-carbon atoms include, but are not limitedto, N, O, S, Si, Al, B, and P. If there is more than one non-carbon atomin the hetero-based parent chain then this atom may be the same elementor may be a combination of different elements, such as N and 0.

The term “mixed ring system” refers to optionally substituted ringstructures that contain at least two rings, and wherein the rings arejoined together by linking, fusing, or a combination thereof. A mixedring system comprises a combination of different ring types, includingcycloalkyl, cycloalkenyl, aryl, and heterocycle.

As used herein, the term “Probiotic Composition” includes a compositioncomprising a probiotic commensal skin bacteria of the disclosure and mayoptionally include compounds described as Formula I or II, that affectsthe microbiome balance of the human skin and which can inhibit cancergrowth, invation and/or metastasis and which can affect pathogen spreadand proliferation. A probiotic composition can comprise an unnaturalratio or composition of an agent or microbe found in nature. Forexample, a microbial probiotic composition can comprise a single type oforganism found on the skin (e.g., S. epidermidis MO34 or MO38 or MO34and MO38) at a cell density or amount not normally found in nature.Alternatively, or in addition, the microbial probiotic composition caninclude a single type of organism as mentioned above, but which ispresent in a composition that does not occur in nature such as a salve,lotion, suspension, ointment and the like. In still another embodiment,a microbial probiotic composition can comprise a microbe at a densitynot normally found in nature or mixed with a non-naturally occurringcomposition at a density not found in nature. In still anotherembodiment, a microbial probiotic composition can comprise arecombinantly engineered microorganism (e.g., an attenuated bacterialspecies). In one embodiment, commensal skin bacteria is a bacteria thatproduces 6-HAP. In another or further embodiment, the baceteriacomprises S. epidermidis MO34 and/or MO38.

The term “purified” and “substantially purified” as used herein refersto cultures, or co-cultures of microorganisms or of biological agent(e.g. fermentation media and extracts, fractionated fermentation media,fermentation by-products, compounds of Formula I or II etc.) that issubstantially free of other cells or components found in the naturalenvironment with which an in vivo-produced agent would naturally beassociated. In some embodiments, a co-culture probiotic can comprise oneor a plurality of commensal skin bacteria.

The term “substituted” with respect to hydrocarbons, heterocycles, andthe like, refers to structures wherein the parent chain contains one ormore substituents. For example, optionally substituted hydrocarbons,hetero-hydrocarbons, heterocycles, mixed ring systems, and the like, caninclude substitution with one or more of the following substituents:halogens, CN, —COOR, —OR, —COR, —OCOOR, —CON(R)₂, —OCON(R)₂, —N(R)₂,NO₂, —SR, —SO₂R, —SO₂N(R)₂ or —SOR groups, wherein R is selected fromthe group comprising a hydrocarbon, a hetero-hydrocarbon, heterocycle,and mixed ring system. Optional substitution of alkyl groups includessubstitution with one or more alkenyl groups, aryl groups or both,wherein the alkenyl groups or aryl groups are also optionallysubstituted. Optional substitution of alkenyl groups includessubstitution with one or more alkyl groups, aryl groups, or both,wherein the alkyl groups or aryl groups are also optionally substituted.Optional substitution of aryl groups includes substitution of the arylring with one or more alkyl groups, alkenyl groups, or both, wherein thealkyl groups or alkenyl groups are also optionally substituted.

The term “substituent” refers to an atom or group of atoms substitutedin place of a hydrogen atom. For purposes of this disclosure, asubstituent would include deuterium atoms.

Optional substituents for hydrocarbons, hetero-hydrocarbons,heterocycles, mixed ring systems, and the like, include among others:

—COOR where R is a hydrogen or an alkyl group or an aryl group and morespecifically where R is methyl, ethyl, propyl, butyl, or phenyl groupsall of which are optionally substituted;

—COR where R is a hydrogen, or an alkyl group or an aryl groups and morespecifically where R is methyl, ethyl, propyl, butyl, or phenyl groupsall of which groups are optionally substituted;

—CON(R)₂ where each R, independently of each other R, is a hydrogen oran alkyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl groups all of which groups areoptionally substituted; R and R can form a ring which may contain one ormore double bonds;

—OCON(R)₂ where each R, independently of each other R, is a hydrogen oran alkyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl groups all of which groups areoptionally substituted; R and R can form a ring which may contain one ormore double bonds;

—N(R)₂ where each R, independently of each other R, is a hydrogen, or analkyl group, acyl group or an aryl group and more specifically where Ris methyl, ethyl, propyl, butyl, or phenyl or acetyl groups all of whichare optionally substituted; or R and R can form a ring which may containone or more double bonds;

—SR, —SO₂R, or —SOR where R is an alkyl group or an aryl groups and morespecifically where R is methyl, ethyl, propyl, butyl, phenyl groups allof which are optionally substituted; for example —SR, R can be hydrogen;

—OCOOR where R is an alkyl group or an aryl groups;

—SO₂N(R)₂ where R is a hydrogen, an alkyl group, or an aryl group and Rand R can form a ring; and

—OR where R═H, alkyl, aryl, or acyl; for example, R can be an acylyielding —OCOR* where R* is a hydrogen or an alkyl group or an arylgroup and more specifically where R* is methyl, ethyl, propyl, butyl, orphenyl groups all of which groups are optionally substituted.

As used herein, the term “Topical” can include administration to theskin externally, as well as shallow injection (e.g., intradermally andintralesionally as described in the Examples) such that a topicalprobiotic composition described herein comes in direct contact withskin.

The term “unsubstituted” with respect to hydrocarbons, heterocycles, andthe like, refers to structures wherein the parent chain contains nosubstituents.

As used herein, a wavy line intersecting another line that is connectedto an atom indicates that this atom is covalently bonded to anotherentity that is present but not being depicted in the structure. A wavyline that does not intersect a line but is connected to an atomindicates that this atom is interacting with another atom by a bond orsome other type of identifiable association.

A bond indicated by a straight line and a dashed line indicates that thebond may be a single covalent bond or alternatively a double covalentbond. But in the case where a ring atom's maximum valence would beexceeded by forming a double covalent bond with another ring atom, thenthe bond would be a single covalent bond.

For the purposes of this disclosure, in the instance that a ring atomdesignated as X would exceed its maximum valence by binding a groupdesignated by R, then the group designated by R would be absent.

Mammalian skin harbors diverse microbial communities whose growth isinfluenced by ecological factors on the body surface such as humidity,temperature, pH, lipid content, and the presence of antimicrobialsproduced by the host. Although the specific mechanisms through whichskin surface microbes influence host function are incompletelyunderstood, specific strains of coagulase-negative staphylococcalspecies have been shown to produce proteins that work together withendogenous host antimicrobial peptides (AMPs) to provide directprotection against infectious pathogens. For example, the production ofphenol-soluble modulins (PSMg and PSMd) by S. epidermidis canselectively kill bacterial pathogens such as S. aureus and group AStreptococcus (GAS). This species has also been shown to benefit skinimmune function by diminishing inflammation after injury, enhancingdevelopment of cutaneous T cells and promoting expression of host AMPssuch as cathelicidins and b-defensins. Germ-free mice are moresusceptible to skin infection than mice maintained under specificpathogen-free conditions or mono associated with S. epidermidis.

Further evidence that commensal Staphylococcus species provide hostdefense has come from observations that nasal colonization with either aspecific strain of S. epidermidis that produces a serine protease or astrain of Staphylococcus lugdunensis that produces a thiazolidinecontaining cyclic peptide can inhibit nasal colonization by S. aureus.More recently, several strains of S. epidermidis, S. hominis, and othercoagulase-negative staphylococcal species that produce a variety ofpreviously unknown AMPs were found to be deficient in atopic dermatitispatients colonized by S. aureus, and a clinical trial evaluating theeffect of reintroduction of these strains demonstrated that theydirectly reduced S. aureus colonization on humans. Thus, evidence isincreasing that the skin microbiome has an important role in promotinghost defense.

These observations suggest that the skin microbiome may contribute toaspects of host defense. The disclosure describes the molecular analysisof the metabolic products of human skin commensal bacteria. Unexpectedlyit was found that S. epidermidis strain MO34 and MO38 produce anucleobase analog with the capacity to inhibit DNA synthesis. Whenadministered intravenously or topically applied to mice, this moleculeor the live S. epidermidis strain(s) itself suppressed tumor growth invivo.

The disclosure provides a composition for treating cancer and/or apathogen infection comprising a compound of formula I(a), I(b) and/orII, alone or in combination with (e.g., produced by) a commensalprobiotic composition comprising an S. epidermidis strain that producesa compound of the disclosure. In one embodiment, the disclosure providesa method and composition comprising 6-HAP. In one embodiment, thecomposition and method comprise a probiotic commensal bacterial theproduces 6-HAP. In another embodiment, the composition and methodcomprise a substantially purified 6-HAP or analog or derivative thereof.In another or further embodiment, the composition comprising 6-HAPcomprises a commensal probiotic and a purified 6-HAP or analog orderivative thereof.

The disclosure demonstrates that compounds comprising a structure ofFormula I(a), Formula I(b) and/or Formula II have the ability to inhibitreplication and expression of DNA. For example, the disclosuredemonstrates that a compound for Formula II (6-HAP) has important andunique host defense capabilities. 6-HAP suppressed growth of major skinpathogens such as GAS, GBS, S. aureus (including MRSA) and P.aeruginosa. Importantly, this antimicrobial activity was selective forthese skin pathogens over human skin commensals such as S. epidermidis,S. hominis and P. acnes. Thus, the disclosure provides methods andcompositions useful for treating infections by contacting a pathogenwith a compound of Formula I(a), I(b) and/or II alone or in combinationwith a probiotic commensal bacterial fo the disclosure.

In addition, an unexpected discovery was made while studying themechanism of action of 6-HAP that led to the identification that thismolecule also has selective anti-proliferative function againstmammalian tumor cell lines and UV-induced skin tumor. As describedelsewhere herein, the 6-HAP compound or derivatives thereof (e.g.,Formula I(a), and I(b)) as well as probiotic commensal bacterial of thedisclosure that can produce 6-HAP can be used to treat neoplasm andcancer.

The disclosure demonstrates that 6-HAP did not exert activity throughdisruption of cell membranes. 6-HAP directly inhibited adenine-thymidinebase pair matching in a cell free assay. Thus, the mechanism of actionof 6-HAP is through inhibition of DNA synthesis. In 6-HAP, the aminogroup at the carbon C-6 position of the purine ring is replaced with ahydroxyamino group. This is a critical position for DNA synthesis sincethe hydrogen of the amino group at the carbon C-6 position of adenine isrequired to bind with oxygen at the carbon C-4 position of thymine.

Commensal skin microbes have not previously been shown to producenucleobase analogs with such activity. However, the capacity of otherchemically-synthesized nucleobase analogs to inhibit DNA synthesis isknown. For example, 6-mercaptopurine is converted in vivo to6-thioguanine and is then incorporated into DNA in place of guanine.8-Azaguanine also suppresses DNA synthesis by a similar mechanism.Similarly to 6-HAP, 6-thioguanine and 8-azaguanine have both antibioticand antineoplastic activities. It is highly unlikely that a commoncommensal produces a potent mutagen that would not be previouslydetected. However, if this was indeed the case, the current observationswould remain highly significant since this would identify a previouslyundetected risk factor for cancer. Thus, this observation of thecapacity of a commensal skin microbe to produce a nucleobase analog ishighly significant.

A remarkable quality of 6-HAP as a nucleobase analog is the capacity toexert selective activity against pathogenic bacteria and tumor celllines, but little toxicity to commensals or normal cells. Previouslyidentified AMPs from S. epidermidis or S. hominis were also known toexert selective killing, a logical behavior if the host cell is toresist killing itself. The mechanism responsible for selective killingby these AMPs is poorly understood but thought to be due to differencesin the capacity to disrupt the cell membrane. In the case of 6-HAP, onlysome pathogens and cancer cell lines were inhibited in vitro and invivo. No systemic toxicity of 6-HAP was observed when mice wererepeatedly administrated this intravenously, nor were growth of normalkeratinocytes inhibited by high concentrations of 6-HAP in culture. Incontrast, the DNA synthesis inhibitor mitomycin C did not show suchselective effects on cell growth. The evidence therefore suggest thatthe selectivity by 6-HAP is not simply due to the rate of cell division.6-HAP exhibits no toxicity to a wild-type strain of E. coli, whereas itinhibited growth of mutant strains deficient in genes involved inmolybdenum cofactors. The data herein demonstrate that the molybdoenzymemARC2 protected NHEKs from 6-HAP. In addition, relative expression levelof mARC2 was higher in NHEKs than cancer cell lines. Moreover, thedisclosure demonstrates the involvement of molybdoenzymes that may becapable of detoxifying 6-HAP to enable selective activity. Thus, in oneembodiment, the disclosure provides a method of treating an infection orcancer, wherein the pathogen of the infection or the cancer cell hasmARC2 expression at a level that is less than normal healthy cells ofthe subject, the method comprising administering a probiotic of thedisclosure and/or a compound of formula I(a), I(b) and/or II to thepathogen or cancer cell.

The disclosure thus provides an entirely new concept that some membersof our skin microbiome may suppress tumor growth and UV-induced tumorformation. Most prior observations have reported that dysbiosis (a stateof altered microbiome) can promote cancer. Observations associatingbacteria in the gut with an increase in carcinogenesis suggested thiseffect was dependent on inflammation. Intestinal inflammation has alsobeen reported to promote development of tumors through increasing thecapacity of microbiota to produce genotoxins which elicit DNA damage.

The importance of this disclosure are several fold. The selectiveactivity of 6-HAP may be essential for maintaining homeostasis of theskin microbiome and could be exploited therapeutically to treat S.aureus infection or colonization, which plays important role inpathogenesis of atopic dermatitis, as well as in treating cancerprogression or skin damage leading to cancer. Such a strategy fordefense is theoretically superior to the use of existing pharmaceuticalantibiotics or antiseptics that non-specifically kill beneficialcommensal bacteria and disrupt homeostasis by killing the normalmicroflora. In addition, long-lasting protection could be achieved ifthe applied beneficial bacteria could successfully colonize on the skinsurface. Further, the disclosure shows the surprising presence ofanti-neoplastic activity from S. epidermidis. The observation that abacterial product can directly limit tumor growth suggests a paradigmshift in the understanding of the functions of the human skinmicrobiome.

The disclosure thus provides a method of treating or reducing the riskof skin infection and/or cancer (e.g., skin cancer) by promoting aneffective skin biome comprising S. epidermidis that produces 6-HAP. Thedisclosure also provides a method of treating or reducing the risk ofinfection and/or skin cancer comprising administering a probioticcomprising S. epidermidis that produces an anticancer agent of thedisclosure. In one embodiment, the probiotic composition comprises S.epidermidis M034 and/or M038 or attenuated or genetically engineeredstrains thereof.

The disclosure provides a probiotic composition for inhibiting and/ormodulating skin damage and neoplasms, more particularly of the skin, andpreferably human skin. In particular embodiment, the probioticcomposition of the disclosure comprises a commensal strain ofStaphylococcus epidermidis that protects against skin neoplasia byproducing the compound 6-N-hydroxyaminopurine (6-HAP). In oneembodiment, the straing is S. epidermidis M034 and/or M038.

The probiotic compositions of the disclosure can be used to treatneoplastic diseases and disorders, improve healing and reduce morbidityassociated with skin damage and neoplasms as well as treating infectionthrough the antimicrobial activity of 6-HAP. For example, a topicalprobiotic compositions can be used to treat a skin damage caused by UVirradiation by contacting the skin with a therapeutically effectiveamount or inhibitive effective amount of a composition as describedbelow and herein. The composition can comprise one or more of an S.epidermidis M034 and/or M035 alone or in combination with 6-HAP or the6-HAP or a derivative thereof alone and any other desired activeingredients that improves skin health.

Any of a variety of methods known in the art can be used to administer aprobiotic composition or compound of the disclosure to a subject. Forexample, a 6-HAP anti-neoplastic agent and/or microbial probioticcomposition of the disclosure may be formulated for topicaladministration (e.g., as a lotion, cream, spray, gel, or ointment). Suchtopical formulations are useful in treating or inhibiting neoplasticcells, UV damage leading to neoplasms and the like. Examples offormulations include topical lotions, creams, soaps, wipes, and thelike. In embodiments, where a neoplasia or cancer is not topical, theadministration of a compound of formula I(a), I(b) and/or II can bedelivered intraperitoneally, intravenously, by inhalation etc.

The disclosure thus provides a method of treating or reducing the riskof cancer (e.g., skin cancer) by promoting an effective skin biomecomprising S. epidermidis. The disclosure also provides a method oftreating or reducing the risk of skin cancer comprising administering aprobiotic comprising S. epidermidis that produces an anticancer agent ofthe disclosure.

The disclosure also provides an antimicrobial/anticancer molecule havingthe general formula of Formula I(a):

or a pharmaceutically acceptable salt or prodrug thereof, wherein,

N¹-N⁵ are nitrogen atoms;

X¹-X² are carbon atoms;

the R groups attached by a dashed line are present, or are not presentif the R group is connected to an atom that is bound to another atom bya double covalent bond;

the bond indicated by both a straight line and a dashed line indicatethat the bond may be a single covalent bond or a double covalent bond;

the fused heterocyclic ring system comprises three double bonds with N²or N³ forming a double bond and with X¹, and with N⁴ or N⁵ forming adouble bond with X²;

R¹ is a hydroxyl, ester, carboxylic acid, or —O—R¹⁰;

R², R⁴, R⁵, R⁷-R⁹ are independently a H, D, optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-alkenyl, optionallysubstituted (C₁-C₆)-alkynyl, optionally substituted (C₃-C₁₂)cycloalkyl,optionally substituted (C₄-C₁₂)cycloalkenyl, optionally substitutedaryl;

R³ and R⁶ are independently selected from a H, D, optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-heteroalkyl, optionallysubstituted (C₁-C₆)-alkenyl, optionally substituted(C₁-C₆)-heteroalkenyl, optionally substituted (C₁-C₆)-alkynyl,optionally substituted (C₁-C₆)-heteroalkynyl, optionally substituted(C₃-C₁₂)cycloalkyl, optionally substituted (C₄-C₁₂)cycloalkenyl,optionally substituted aryl, optionally substituted heterocycle, halide,hydroxyl, carbonyl, aldehyde, carboxyl, ester, alkoxy, carboxyamide,amine, imine, azide, cyano, nitro, nitroso, thiol, sulfide, sulfoxide,sulfone, and phosphate;

R¹⁰ is selected from D, optionally substituted (C₁-C₆)-alkyl, optionallysubstituted (C₁-C₆)-heteroalkyl, optionally substituted (C₁-C₆)-alkenyl,optionally substituted (C₁-C₆)-heteroalkenyl, optionally substituted(C₁-C₆)-alkynyl, optionally substituted (C₁-C₆)-heteroalkynyl,optionally substituted (C₃-C₁₂)cycloalkyl, optionally substituted(C₄-C₁₂)cycloalkenyl, optionally substituted aryl, and optionallysubstituted heterocycle. In another embodiment, the compound has thegeneral formulat of Formulat I(b):

or a pharmaceutically acceptable salt or prodrug thereof, wherein,

N¹-N⁵ are nitrogen atoms;

X¹-X² are carbon atoms;

the R groups attached by a dashed line are present, or are not presentif the R group is connected to an atom that is bound to another atom bya double covalent bond;

the bond indicated by both a straight line and a dashed line indicatethat the bond may be a single covalent bond or a double covalent bond;

the fused heterocyclic ring system comprises three double bonds with N²or N³ forming a double bond with X¹, and with N⁴ or N⁵ forming a doublebond with X²;

R¹ is a hydroxyl, ester, carboxylic acid, or —O—R¹⁰;

R², R⁴, R⁵, and R⁷ are independently a H, D, optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-alkenyl, optionallysubstituted (C₁-C₆)-alkynyl, optionally substituted (C₃-C₁₂)cycloalkyl,optionally substituted (C₄-C₁₂)cycloalkenyl, optionally substitutedaryl;

R¹⁰ is selected from D, optionally substituted (C₁-C₆)-alkyl, optionallysubstituted (C₁-C₆)-heteroalkyl, optionally substituted (C₁-C₆)-alkenyl,optionally substituted (C₁-C₆)-heteroalkenyl, optionally substituted(C₁-C₆)-alkynyl, optionally substituted (C₁-C₆)-heteroalkynyl,optionally substituted (C₃-C₁₂)cycloalkyl, optionally substituted(C₄-C₁₂)cycloalkenyl, optionally substituted aryl, and optionallysubstituted heterocycle.

In yet another embodiment, the disclosure provides a compound of generalformula II:

or a pharmaceutically acceptable salt or prodrug thereof; or a tautomerof the compound of Formula II, or a pharmaceutically acceptable salt orprodrug of the tautomer of compound of Formula II thereof.

Methods and compositions useful for treatment of cancer are provided. Inone embodiment the disclosure provides compositions and methods usefulfor treating a cancer wherein the methods and compositions compriseFormula I(a), I(b) and/or II, a derivative or salt thereof. The methodsand compositions of the disclosure can be used alone or in combinationwith other anticancer agents to treat such cancer. In one embodiment,the composition comprises S. epidermidis MO34 and/or MO38 in addition toa compound of Formula I(a), I(b) and/or II.

Suitable acids for use in the preparation of pharmaceutically acceptablesalts of a compound of the disclosure, include, but are not limited to,aceptic acid, 2,2-dichoroacetic acid, acylated amino acids, adipic acid,alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid,benzoic acid, 4-acetamidobenzoic acid, boric acid, (+)-camphoric acid,camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid,caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid,cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,D-gluconic acid, D-glucuronic acid, L-glutamic acid, α-oxo-glutaricacid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid,hydroiodic acid, (+)-L-lactic acid, (+/−)-DL-lactic acid, lactobionicacid, lauric acid, maleic acid, (−)-L-malic acid, malonic acid,(+/−)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonicacid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphtoic acid,nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid,palmitic acid, pamoic acid, perchloric acid, phosphoric acid,L-pyroglutamic acid, saccharic acid, salicyclic acid, 4-amino-salicylicacid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannicacid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid,undecylenic acid, and valeric acid.

Suitable acids for use in the preparation of pharmaceutically acceptablesalts, include, but are not limited to, inorganic bases, such asmagnesium hydroxide, calcium hydroxide, potassium hydroxide, zinchydroxide, or sodium hydroxide; and organic bases, such as primary,secondary, tertiary, and quaternary, aliphatic and aromatic amines,including L-arginine, benethamine, benzathine, choline, deanol,diethanolamine, diethylamine, dimethylamine, dipropylamine,diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylamine,ethylenediamine, isopropylamine, N-methyl-glucamine, hydrabamine,1H-imidazole, L-Lysine, morpholine, 4-(2-hydroxyethyl)-morpholine,methylamine, piperidine, piperazine, propylamine, pyrrolidine,1-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline,isoquinoline, secondary amines, triethanolamine, trimethylamine,triethylamine, N-methyl-D-glucamine,2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine.

Pharmaceutically acceptable salts comprise pharmaceutically-acceptableanions and/or cations. Pharmaceutically-acceptable cations include amongothers, alkali metal cations (e.g., Li+, Na+, K+), alkaline earth metalcations (e.g., Ca²⁺, Mg²⁺), non-toxic heavy metal cations and ammonium(NH₄ ⁺) and substituted ammonium (N(R′)₄ ⁺, where R′ is hydrogen, alkyl,or substituted alkyl, i.e., including, methyl, ethyl, or hydroxyethyl,specifically, trimethyl ammonium, triethyl ammonium, and triethanolammonium cations). Pharmaceutically-acceptable anions include amongother halides (e.g., Cl—, Br—), sulfate, acetates (e.g., acetate,trifluoroacetate), ascorbates, aspartates, benzoates, citrates, andlactate.

A compound disclosed herein may also have a prodrug form. A prodrug is afunctional derivative of the compound disclosed herein and is readilyconvertible into the parent compound in vivo. Prodrugs are often usefulbecause, in some situations, they may be easier to administer than theparent compound. They may, for instance, be bio-available by oraladministration whereas the parent compound is not. The prodrug may alsohave enhanced solubility in pharmaceutical compositions over the parentcompound. A prodrug may be converted into the parent drug by variousmechanisms, including enzymatic processes and metabolic hydrolysis.Various examples and forms of prodrugs are well known in the art.Examples of prodrugs are found, inter alia, in Design of Prodrugs,edited by H. Bundgaard, (Elsevier, 1985), Methods in Enzymology, Vol.42, at pp. 309-396, edited by K. Widder, et. al. (Academic Press, 1985);A Textbook of Drug Design and Development, edited by Krosgaard-Larsenand H. Bundgaard, Chapter 5, “Design and Application of Prodrugs,” by H.Bundgaard, at pp. 113-191, 1991); H. Bundgaard, Advanced Drug DeliveryReviews, Vol. 8, p. 1-38 (1992); H. Bundgaard, et al., Journal ofPharmaceutical Sciences, Vol. 77, p. 285 (1988); and Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392). In a specific example, if a parent compounddisclosed herein has a hydroxyl group, this hydroxyl group may beconverted to an ester in attempts to increase bioavailability,solubility, injection site pain relief, elimination of an unpleasanttaste, decreased toxicity, decreased metabolic inactivation, increasedchemical stability, and/or prolonged or shortened action of the hydroxylcontaining parent compound. In another specific example, if a parentcompound disclosed herein has an amine group, this amine group may beconverted to a Schiff base in attempts to increase bioavailability,solubility, injection site pain relief, elimination of an unpleasanttaste, decreased toxicity, decreased metabolic inactivation, increasedchemical stability, and/or prolonged or shortened action of the hydroxylcontaining parent compound.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, can be in a form suitable for administration to a subjectusing carriers, excipients, and additives or auxiliaries. Frequentlyused carriers or auxiliaries include magnesium carbonate, titaniumdioxide, lactose, mannitol and other sugars, talc, milk protein,gelatin, starch, vitamins, cellulose and its derivatives, animal andvegetable oils, polyethylene glycols and solvents, such as sterilewater, alcohols, glycerol, and polyhydric alcohols. Intravenous vehiclesinclude fluid and nutrient replenishers. Preservatives includeantimicrobial, chelating agents, and inert gases. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in Remington's Pharmaceutical Sciences, 15th ed., Easton: MackPublishing Co., 1405-1412, 1461-1487 (1975), and The National FormularyXIV., 14th ed., Washington: American Pharmaceutical Association (1975),the contents of which are hereby incorporated by reference. The pH andexact concentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's, The Pharmacological Basis for Therapeutics (7thed.).

A pharmaceutical composition comprising a probiotic disclosed hereincomprising a commensal bacterial (e.g., S. epidermidis M034 and/or M038)or an engineered form thereof (e.g., attenuated or geneticallymodified), may be formulated in any dosage form that is suitable fortopical administration for local or systemic effect, includingemulsions, solutions, suspensions, creams, gels, hydrogels, ointments,dusting powders, dressings, elixirs, lotions, suspensions, tinctures,pastes, foams, films, aerosols, irrigations, sprays, suppositories,bandages, dermal patches. The topical formulation comprising a probioticdisclosed herein may also comprise liposomes, micelles, microspheres,nanosystems, and mixtures thereof.

In one embodiment, a bandage or dressing is provided comprising acompound of formula I(a), I(b) and/or II, and/or a probiotic commensalskin bacteria described herein. In various aspects, a bandage ordressing is provided the major constituents of which includes a matrixand a topical probiotic composition of Formulas I or II described above.In various aspects, a bandage or dressing is provided the majorconstituents of which includes a matrix and a probiotic commensal skinbacteria. In various aspects, a bandage or dressing is provided themajor constituents of which includes a matrix and a probiotic commensalskin bacteria fermentation extract. In various aspects, a bandage ordressing is provided the major constituents of which includes a matrixand glycerol. In one embodiment, the bandage or dressing is applied tosite of skin damage or injury. In another embodiment, the bandage ordressing is applied to a site of infection.

A “pharmaceutically acceptable carrier” is intended to include solvents,dispersion media, coatings, antibacterial and antifungal agents (asneeded so long as they are not detrimental to the probiotic commensalbacteria), isotonic and absorption delaying agents, and the like. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the pharmaceutical composition, use thereof in thetherapeutic compositions and methods of treatment is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Pharmaceutically acceptable carriers and excipients suitable for use inthe topical formulations disclosed herein include, but are not limitedto, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles,stabilizers, solubility enhancers, isotonic agents, buffering agents,antioxidants, local anesthetics, suspending and dispersing agents,wetting or emulsifying agents, complexing agents, sequestering orchelating agents, penetration enhancers, cryopretectants,lyoprotectants, thickening agents, and inert gases.

A pharmaceutical composition comprising a probiotic may be formulated inthe forms of ointments, creams, sprays and gels. Suitable ointmentvehicles include oleaginous or hydrocarbon vehicles, including such aslard, benzoinated lard, olive oil, cottonseed oil, and other oils, whitepetrolatum; emulsifiable or absorption vehicles, such as hydrophilicpetrolatum, hydroxystearin sulfate, and anhydrous lanolin;water-removable vehicles, such as hydrophilic ointment; water-solubleointment vehicles, including polyethylene glycols of varying molecularweight; emulsion vehicles, either water-in-oil (W/O) emulsions oroil-in-water (O/W) emulsions, including cetyl alcohol, glycerylmonostearate, lanolin, and stearic acid (see, Remington: The Science andPractice of Pharmacy). These vehicles are emollient but generallyrequire addition of antioxidants and preservatives.

Suitable cream base can be oil-in-water or water-in-oil. Cream vehiclesmay be water-washable, and contain an oil phase, an emulsifier, and anaqueous phase. The oil phase is also called the “internal” phase, whichis generally comprised of petrolatum and a fatty alcohol such as cetylor stearyl alcohol. The aqueous phase usually, although not necessarily,exceeds the oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation may be a nonionic, anionic, cationic,or amphoteric surfactant.

Gels are semisolid, suspension-type systems. Single-phase gels containmaterial substantially uniformly throughout the liquid carrier. Suitablegelling agents include crosslinked acrylic acid polymers, such ascarbomers, carboxypolyalkylenes, Carbopol®; hydrophilic polymers, suchas polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, andpolyvinylalcohol; cellulosic polymers, such as hydroxypropyl cellulose,hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropylmethylcellulose phthalate, and methylcellulose; gums, such as tragacanthand xanthan gum; sodium alginate; and gelatin. In order to prepare auniform gel, dispersing agents such as alcohol or glycerin can be added,or the gelling agent can be dispersed by trituration, mechanical mixing,and/or stirring.

A pharmaceutical composition comprising a probiotic disclosed herein,may be administered rectally, urethrally, vaginally, or perivaginally inthe forms of suppositories, pessaries, bougies, poultices or cataplasm,pastes, powders, dressings, creams, plasters, contraceptives, ointments,solutions, emulsions, suspensions, tampons, gels, foams, sprays, orenemas. These dosage forms can be manufactured using conventionalprocesses as described in Remington: The Science and Practice ofPharmacy.

The pharmaceutical compositions according to the disclosure may beadministered locally or systemically. A “therapeutically effective dose”is the quantity of an agent (e.g., a compound of Formula I(a), I(b)and/or II) or a probiotic comprising a commensal bacterial according tothe disclosure necessary to prevent, to cure, or at least partiallyarrest the symptoms of cancer (e.g., proliferation, metastasis, growthetc.). Amounts effective for this use will, of course, depend on theseverity of the cancer, the weight and general state of the subjectand/or the surface area to be treated. Typically, dosages used in vitromay provide useful guidance in the amounts useful for human and animaltreatment. Various considerations are described, e.g., in Langer,Science, 249: 1527, (1990); Gilman et al. (eds.) (1990), each of whichis herein incorporated by reference. Dosage regima can be adjusted toprovide the optimum therapeutic response. For example, several divideddoses can be administered daily or the dose can be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

The principal pharmaceutical composition is compounded for convenientand effective administration in effective amounts with a suitablepharmaceutically acceptable carrier in an acceptable dosage unit. In thecase of compositions containing supplementary active ingredients, thedosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

The disclosure provides for a compound (e.g., a compound of FormulaI(a), I(b) and/or II) disclosed herein, derivative or analog thereof,including pharmaceutical salt forms and prodrug forms, that can beadministered to any host, including a human or non-human animal, in anamount effective to inhibit the growth, spread or proliferation of acancer cell or neoplasm. In one embodiment, the administration resultsin the inhibition of growth, proliferation, migration and/or metastasisof a cancer or neoplastic cell.

Any of a variety of art-known methods can be used to administer acompound (e.g., a compound of Formula I(a), I(b) and/or II) disclosedherein, a derivative or analog thereof, including pharmaceutical saltforms and prodrug forms, either alone or used in combination with one ormore other therapeutic agents. For example, administration can beparenterally by injection or by gradual infusion over time. The agent(s)can be administered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, by inhalation, topically or transdermally.

A pharmaceutical composition (e.g., a compound of Formula I(a), I(b)and/or II or a probiotic composition comprising a commensal bacterialsuch as S. epidermidis MO34 and/or MO38, or a combination of a commensalbacteria and a compound of the disclosure) can be administered in aconvenient and appropriate manner, such as by injection (subcutaneous,intravenous, etc.), oral administration, inhalation, transdermalapplication, topical via lotion, creame or ointment, or rectaladministration. Depending on the route of administration, thepharmaceutical composition can be coated with a material to protect thepharmaceutical composition from the action of enzymes, acids, and othernatural conditions that may inactivate the pharmaceutical composition.The pharmaceutical composition can also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof, and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

In another embodiment, a pharmaceutical composition comprising acompound and/or a commenal probiotic disclosed herein, a derivative oranalog thereof, including pharmaceutical salt forms and prodrug forms,can be formulated either alone or in combination with one or moreadditional therapeutic agents, including, but not limited to,chemotherapeutics, antibiotics (so long as they don't destroy theprobiotic benefits), antifungal-agents, anti-pruritics, analgesics,and/or antiviral agents.

Topical administration, as used herein, include (intra)dermal,conjunctival, intracorneal, intraocular, ophthalmic, auricular,transdermal, nasal, vaginal, uretheral, respiratory, and rectaladministration. Such topical formulations are useful in treating orinhibiting cancers of the eye, skin, and mucous membranes (e.g., mouth,vagina, rectum). Examples of formulations in the market place includetopical lotions, creams, soaps, wipes, and the like.

Rectal, urethral, and vaginal suppositories are solid bodies forinsertion into body orifices, which are solid at ordinary temperaturesbut melt or soften at body temperature to release the activeingredient(s) inside the orifices. Pharmaceutically acceptable carriersutilized in rectal and vaginal suppositories include bases or vehicles,such as stiffening agents, which produce a melting point in theproximity of body temperature, when formulated with the pharmaceuticalcompositions disclosed herein; and antioxidants as described herein,including bisulfite and sodium metabisulfite. Suitable vehicles include,but are not limited to, cocoa butter (theobroma oil), glycerin-gelatin,carbowax (polyoxyethylene glycol), spermaceti, paraffin, white andyellow wax, and appropriate mixtures of mono-, di- and triglycerides offatty acids, hydrogels, such as polyvinyl alcohol, hydroxyethylmethacrylate, polyacrylic acid; glycerinated gelatin. Combinations ofthe various vehicles may be used. Rectal and vaginal suppositories maybe prepared by the compressed method or molding. The typical weight of arectal and vaginal suppository is about 2 to about 3 g.

Solutions or suspensions for use in a pressurized container, pump,spray, atomizer, or nebulizer may be formulated to contain ethanol,aqueous ethanol, or a suitable alternative agent for dispersing,solubilizing, or extending release of the active ingredient disclosedherein, a propellant as solvent; and/or a surfactant, such as sorbitantrioleate, oleic acid, or an oligolactic acid.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be micronized to a size suitable for delivery byinhalation, such as about 50 micrometers or less, or about 10micrometers or less. Particles of such sizes may be prepared using acomminuting method known to those skilled in the art, such as spiral jetmilling, fluid bed jet milling, supercritical fluid processing to formnanoparticles, high pressure homogenization, or spray drying.

Capsules, blisters and cartridges for use in an inhaler or insufflatormay be formulated to contain a powder mix of the pharmaceuticalcompositions disclosed herein; a suitable powder base, such as lactoseor starch; and a performance modifier, such as 1-leucine, mannitol, ormagnesium stearate. The lactose may be anhydrous or in the form of themonohydrate. Other suitable excipients or carriers include dextran,glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose. Apharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, for inhaled/intranasal administration may furthercomprise a suitable flavor, such as menthol and levomenthol, orsweeteners, such as saccharin or saccharin sodium.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, for topical administration may be formulated to beimmediate release or modified release, including delayed-, sustained-,pulsed-, controlled-, targeted, and programmed release.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be formulated into liposomes to reduce toxicity orincrease bioavailability. Other methods for delivery include oralmethods that entail encapsulation of the in microspheres or proteinoids,aerosol delivery (e.g., to the lungs), or transdermal delivery (e.g., byiontophoresis or transdermal electroporation). Other methods ofadministration will be known to those skilled in the art.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be formulated as a modified release dosage form. Asused herein, the term “modified release” refers to a dosage form inwhich the rate or place of release of the active ingredient(s) isdifferent from that of an immediate dosage form when administered by thesame route. Modified release dosage forms include delayed-, extended-,prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-,targeted-, programmed-release, and gastric retention dosage forms. Thepharmaceutical compositions in modified release dosage forms can beprepared using a variety of modified release devices and methods knownto those skilled in the art, including, but not limited to, matrixcontrolled release devices, osmotic controlled release devices,multiparticulate controlled release devices, ion-exchange resins,enteric coatings, multilayered coatings, microspheres, liposomes, andcombinations thereof. The release rate of the active ingredient(s) canalso be modified by varying the particle sizes and polymorphorism of theactive ingredient(s). Examples of modified release include, but are notlimited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767;5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566;5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945; 5,993,855;6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970;6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; and 6,699,500.

The pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, that is formulated in a modified release dosage form maybe fabricated using a matrix controlled release device (see, Takada etal in “Encyclopedia of Controlled Drug Delivery,” Vol. 2, Mathiowitzed., Wiley, 1999).

In one embodiment, the pharmaceutical composition comprising a compounddisclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms, in a modified releasedosage form is formulated using an erodible matrix device, which iswater-swellable, erodible, or soluble polymers, including syntheticpolymers, and naturally occurring polymers and derivatives, such aspolysaccharides and proteins.

Materials useful in forming an erodible matrix include, but are notlimited to, chitin, chitosan, dextran, and pullulan; gum agar, gumarabic, gum karaya, locust bean gum, gum tragacanth, carrageenans, gumghatti, guar gum, xanthan gum, and scleroglucan; starches, such asdextrin and maltodextrin; hydrophilic colloids, such as pectin;phosphatides, such as lecithin; alginates; propylene glycol alginate;gelatin; collagen; and cellulosics, such as ethyl cellulose (EC),methylethyl cellulose (MEC), carboxymethyl cellulose (CMC), CMEC,hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), celluloseacetate (CA), cellulose propionate (CP), cellulose butyrate (CB),cellulose acetate butyrate (CAB), CAP, CAT, hydroxypropyl methylcellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetatetrimellitate (HPMCAT), and ethylhydroxy ethylcellulose (EHEC); polyvinylpyrrolidone; polyvinyl alcohol; polyvinyl acetate; glycerol fatty acidesters; polyacrylamide; polyacrylic acid; copolymers of ethacrylic acidor methacrylic acid (EUDRAGIT, Rohm America, Inc., Piscataway, N.J.);poly(2-hydroxyethyl-methacrylate); polylactides; copolymers ofL-glutamic acid and ethyl-L-glutamate; degradable lactic acid-glycolicacid copolymers; poly-D-(−)-3-hydroxybutyric acid; and other acrylicacid derivatives, such as homopolymers and copolymers ofbutylmethacrylate, methylmethacrylate, ethylmethacrylate, ethylacrylate,(2-dimethylaminoethyl)methacrylate, and(trimethylaminoethyl)methacrylate chloride.

In further embodiments, a pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated with anon-erodible matrix device. The active ingredient(s) is dissolved ordispersed in an inert matrix and is released primarily by diffusionthrough the inert matrix once administered. Materials suitable for useas a non-erodible matrix device included, but are not limited to,insoluble plastics, such as polyethylene, polypropylene, polyisoprene,polyisobutylene, polybutadiene, polymethylmethacrylate,polybutylmethacrylate, chlorinated polyethylene, polyvinylchloride,methyl acrylate-methyl methacrylate copolymers, ethylene-vinylacetatecopolymers, ethylene/propylene copolymers, ethylene/ethyl acrylatecopolymers, vinylchloride copolymers with vinyl acetate, vinylidenechloride, ethylene and propylene, ionomer polyethylene terephthalate,butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, polyvinyl chloride, plasticizednylon, plasticized polyethyleneterephthalate, natural rubber, siliconerubbers, polydimethylsiloxanes, silicone carbonate copolymers;hydrophilic polymers, such as ethyl cellulose, cellulose acetate,crospovidone, and cross-linked partially hydrolyzed polyvinyl acetate;and fatty compounds, such as carnauba wax, microcrystalline wax, andtriglycerides.

In a matrix controlled release system, the desired release kinetics canbe controlled, for example, via the polymer type employed, the polymerviscosity, the particle sizes of the polymer and/or the activeingredient(s), the ratio of the active ingredient(s) versus the polymer,and other excipients or carriers in the compositions.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, in a modified release dosage form may be prepared bymethods known to those skilled in the art, including direct compression,dry or wet granulation followed by compression, melt-granulationfollowed by compression.

The pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, in a modified release dosage form may be fabricated usingan osmotic controlled release device, including one-chamber system,two-chamber system, asymmetric membrane technology (AMT), and extrudingcore system (ECS). In general, such devices have at least twocomponents: (a) the core which contains the active ingredient(s) and (b)a semipermeable membrane with at least one delivery port, whichencapsulates the core. The semipermeable membrane controls the influx ofwater to the core from an aqueous environment of use so as to cause drugrelease by extrusion through the delivery port(s).

In addition to the active ingredient(s), the core of the osmotic deviceoptionally includes an osmotic agent, which creates a driving force fortransport of water from the environment of use into the core of thedevice. One class of osmotic agents water-swellable hydrophilicpolymers, which are also referred to as “osmopolymers” and “hydrogels,”including, but not limited to, hydrophilic vinyl and acrylic polymers,polysaccharides such as calcium alginate, polyethylene oxide (PEO),polyethylene glycol (PEG), polypropylene glycol (PPG),poly(2-hydroxyethyl methacrylate), poly(acrylic) acid, poly(methacrylic)acid, polyvinylpyrrolidone (PVP), crosslinked PVP, polyvinyl alcohol(PVA), PVA/PVP copolymers, PVA/PVP copolymers with hydrophobic monomerssuch as methyl methacrylate and vinyl acetate, hydrophilic polyurethanescontaining large PEO blocks, sodium croscarmellose, carrageenan,hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC),hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC) andcarboxyethyl, cellulose (CEC), sodium alginate, polycarbophil, gelatin,xanthan gum, and sodium starch glycolate.

The other class of osmotic agents are osmogens, which are capable ofimbibing water to affect an osmotic pressure gradient across the barrierof the surrounding coating. Suitable osmogens include, but are notlimited to, inorganic salts, such as magnesium sulfate, magnesiumchloride, calcium chloride, sodium chloride, lithium chloride, potassiumsulfate, potassium phosphates, sodium carbonate, sodium sulfite, lithiumsulfate, potassium chloride, and sodium sulfate; sugars, such asdextrose, fructose, glucose, inositol, lactose, maltose, mannitol,raffinose, sorbitol, sucrose, trehalose, and xylitol; organic acids,such as ascorbic acid, benzoic acid, fumaric acid, citric acid, maleicacid, sebacic acid, sorbic acid, adipic acid, edetic acid, glutamicacid, p-tolunesulfonic acid, succinic acid, and tartaric acid; urea; andmixtures thereof.

Osmotic agents of different dissolution rates may be employed toinfluence how rapidly the active ingredient(s) is initially deliveredfrom the dosage form. For example, amorphous sugars, such as MannogemeEZ (SPI Pharma, Lewes, Del.) can be used to provide faster deliveryduring the first couple of hours to promptly produce the desiredtherapeutic effect, and gradually and continually release of theremaining amount to maintain the desired level of therapeutic orprophylactic effect over an extended period of time. In this case, theactive ingredient(s) is released at such a rate to replace the amount ofthe active ingredient metabolized and excreted.

The core may also include a wide variety of other excipients andcarriers as described herein to enhance the performance of the dosageform or to promote stability or processing.

The total amount of the active ingredient(s) released and the releaserate can substantially by modulated via the thickness and porosity ofthe semipermeable membrane, the composition of the core, and the number,size, and position of the delivery ports.

The pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, in an osmotic controlled-release dosage form may furthercomprise additional conventional excipients or carriers as describedherein to promote performance or processing of the formulation.

The osmotic controlled-release dosage forms can be prepared according toconventional methods and techniques known to those skilled in the art(see, Remington: The Science and Practice of Pharmacy, supra; Santus andBaker, J. Controlled Release 1995, 35, 1-21; Verma et al., DrugDevelopment and Industrial Pharmacy 2000, 26, 695-708; Verma et al., J.Controlled Release 2002, 79, 7-27).

In a certain embodiment, a pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated as AMTcontrolled-release dosage form, which comprises an asymmetric osmoticmembrane that coats a core comprising the active ingredient(s) and otherpharmaceutically acceptable excipients or carriers. See, U.S. Pat. No.5,612,059 and WO 2002/17918. The AMT controlled-release dosage forms canbe prepared according to conventional methods and techniques known tothose skilled in the art, including direct compression, dry granulation,wet granulation, and a dip-coating method.

In a particular embodiment, a pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated as ESCcontrolled-release dosage form, which comprises an osmotic membrane thatcoats a core comprising the active ingredient(s), a hydroxylethylcellulose, and other pharmaceutically acceptable excipients or carriers.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be administered parenterally by injection, infusion,or implantation, for local or systemic administration. Parenteraladministration, as used herein, include intravenous, intraarterial,intraperitoneal, intrathecal, intraventricular, intraurethral,intrastemal, intracranial, intramuscular, intrasynovial, andsubcutaneous administration.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be formulated in any dosage forms that are suitablefor parenteral administration, including solutions, suspensions,emulsions, micelles, liposomes, microspheres, nanosystems, and solidforms suitable for solutions or suspensions in liquid prior toinjection. Such dosage forms can be prepared according to conventionalmethods known to those skilled in the art of pharmaceutical science(see, Remington: The Science and Practice of Pharmacy, supra).

The pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may include one or more pharmaceutically acceptablecarriers and excipients, including, but not limited to, aqueousvehicles, water-miscible vehicles, non-aqueous vehicles, antibacterialagents or preservatives against the growth of microorganisms,stabilizers, solubility enhancers, isotonic agents, buffering agents,antioxidants, local anesthetics, suspending and dispersing agents,wetting or emulsifying agents, complexing agents, sequestering orchelating agents, cryoprotectants, lyoprotectants, thickening agents, pHadjusting agents, and inert gases.

Preparations for parenteral administration of a pharmaceuticalcomposition comprising a compound disclosed herein, a derivative oranalog thereof, including pharmaceutical salt forms and prodrug formsinclude sterile aqueous or non-aqueous solutions, suspensions, andemulsions. Suitable aqueous vehicles include, but are not limited to,water, saline, physiological saline or phosphate buffered saline (PBS),sodium chloride injection, Ringers injection, isotonic dextroseinjection, sterile water injection, dextrose, lactated Ringersinjection, alcoholic/aqueous solutions, and emulsions or suspensions.Non-aqueous vehicles include, but are not limited to, injectable organicesters such as ethyl oleate, and fixed oils of vegetable origin, castoroil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil,safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils,hydrogenated soybean oil, and medium-chain triglycerides of coconut oil,palm seed oil. Water-miscible vehicles include, but are not limited to,ethanol, 1,3-butanediol, liquid polyethylene glycol (e.g., polyethyleneglycol 300 and polyethylene glycol 400), propylene glycol, glycerin,N-methyl-2-pyrrolidone, dimethylacetamide, and dimethylsulfoxide.Examples of parenteral vehicles include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, andfixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives such as,other antibacterials, anti-oxidants, cheating agents, inert gases andthe like also can be included.

Suitable antibacterial agents or preservatives that can be used with acompound of the disclosure include, but are not limited to, phenols,cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzates, thimerosal, benzalkonium chloride, benzethoniumchloride, methyl- and propyl-parabens, and sorbic acid. Suitableisotonic agents include, but are not limited to, sodium chloride,glycerin, and dextrose. Suitable buffering agents include, but are notlimited to, phosphate and citrate. Suitable antioxidants are those asdescribed herein, including bisulfate and sodium metabisulfite. Suitablelocal anesthetics include, but are not limited to, procainehydrochloride. Suitable suspending and dispersing agents are those asdescribed herein, including sodium carboxymethylcelluose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Suitable emulsifying agentsinclude those described herein, including polyoxyethylene sorbitanmonolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamineoleate. Suitable sequestering or chelating agents include, but are notlimited to EDTA. Suitable pH adjusting agents include, but are notlimited to, sodium hydroxide, hydrochloric acid, citric acid, and lacticacid. Suitable complexing agents include, but are not limited to,cyclodextrins, including α-cyclodextrin, β-cyclodextrin,hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, andsulfobutylether 7-β-cyclodextrin (CAPTISOL®, CyDex, Lenexa, Kans.).

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms may be formulated for single or multiple dosageadministration. The single dosage formulations are packaged in anampule, a vial, or a syringe. The multiple dosage parenteralformulations must contain an antimicrobial agent at bacteriostatic orfungistatic concentrations. Parenteral formulations are sterile, asknown and practiced in the art.

In one embodiment, the pharmaceutical composition comprising a compounddisclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated asready-to-use sterile solutions. In another embodiment, thepharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms are formulated as sterile dry soluble products, includinglyophilized powders and hypodermic tablets, to be reconstituted with avehicle prior to use. In yet another embodiment, the pharmaceuticalcomposition comprising a compound disclosed herein, a derivative oranalog thereof, including pharmaceutical salt forms and prodrug formsare formulated as ready-to-use sterile suspensions. In yet anotherembodiment, the pharmaceutical composition comprising a compounddisclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated as steriledry insoluble products to be reconstituted with a vehicle prior to use.In still another embodiment, the pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated asready-to-use sterile emulsions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form” as used herein, refers to physically discrete unitssuited as unitary dosages for the individual to be treated; each unitcontaining a predetermined quantity of pharmaceutical composition iscalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the dosageunit forms of the disclosure are related to the characteristics of thepharmaceutical composition and the particular therapeutic effect to beachieve.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, suitable for injectable use include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms, should be sterile andshould be fluid to the extent that easy syringability exists. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size, in the case of dispersion, and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be typical to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating apharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug form in the required amount in an appropriate solvent with oneor a combination of ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating a pharmaceutical composition comprising a compounddisclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms, into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, can be orally administered, for example, with an inertdiluent or an assimilable edible carrier. The pharmaceutical compositioncomprising a compound disclosed herein, a derivative or analog thereof,including pharmaceutical salt forms and prodrug forms, and otheringredients can also be enclosed in a hard or soft-shell gelatincapsule, compressed into tablets, or incorporated directly into theindividual's diet. For oral therapeutic administration, thepharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, can be incorporated with excipients and used in the formof ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations can, of course, bevaried and can conveniently be between about 5% to about 80% of theweight of the unit.

The tablets, troches, pills, capsules, and the like can also contain thefollowing: a binder, such as gum gragacanth, acacia, corn starch, orgelatin; excipients such as dicalcium phosphate; a disintegrating agent,such as corn starch, potato starch, alginic acid, and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin, or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above type, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac, sugar, or both.A syrup or elixir can contain the agent, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye, and flavoring, suchas cherry or orange flavor. Of course, any material used in preparingany dosage unit form should be pharmaceutically pure and substantiallynon-toxic/biocompatible in the amounts employed.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms are disclosed herein, may be formulated as immediate ormodified release dosage forms, including delayed-, sustained-, pulsed-,controlled, targeted-, and programmed-release forms.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms are disclosed herein, may be formulated as a suspension,solid, semi-solid, or thixotropic liquid, for administration as animplanted depot. In one embodiment, the pharmaceutical compositioncomprising a compound disclosed herein, a derivative or analog thereof,including pharmaceutical salt forms and prodrug forms are dispersed in asolid inner matrix, which is surrounded by an outer polymeric membranethat is insoluble in body fluids but allows the active ingredient in thepharmaceutical compositions to diffuse through.

Suitable inner matrixes include polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers, such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol, and cross-linked partiallyhydrolyzed polyvinyl acetate.

Suitable outer polymeric membranes include polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer.

A therapeutically effective amount can be measured as the amountsufficient to decrease a subject's symptoms (e.g., tumor growth, cancerspread and the like). Typically, the subject is treated with an amountof a therapeutic composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, sufficient to reduce a symptom of a disease or disorderby at least 50%, 90% or 100%. Generally, the optimal dosage will dependupon the disorder and factors such as the weight of the subject, thetype of cancer or neoplasm, the weight, sex, and degree of symptoms.Nonetheless, suitable dosages can readily be determined by one skilledin the art. Typically, a suitable dosage is 0.5 to 40 mg/kg body weight,e.g., 1 to 8 mg/kg body weight.

The compounds disclosed herein may also be combined or used incombination with other agents useful in the treatment, prevention, oramelioration of one or more symptoms of various syndromes, disorders,and/or diseases. Or, by way of example only, the therapeuticeffectiveness of one of the compounds described herein may be enhancedby administration of an adjuvant (i.e., by itself the adjuvant may onlyhave minimal therapeutic benefit, but in combination with anothertherapeutic agent, the overall therapeutic benefit to the patient isenhanced).

Such other agents, adjuvants, or drugs, may be administered, by a routeand in an amount commonly used therefor, simultaneously (at the sametime or in the same formulation) or sequentially with a compound asdisclosed herein. When a compound as disclosed herein disclosed hereinis used contemporaneously with one or more other drugs, a pharmaceuticalcomposition containing such other drugs in addition to the compounddisclosed herein may be utilized, but is not required. Accordingly, thepharmaceutical compositions disclosed herein include those that alsocontain one or more other active ingredients or therapeutic agents(e.g., a chemotherapeutic or other anti-cancer agent, an antibiotic, andthe like), in addition to a compound disclosed herein.

Examples of chemotherapeutic agents include: alkylating agents such asthiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and tiimethylolomelamine; acetogenins(e.g., bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; vinca alkaloids; epipodophyllotoxins; antibiotics suchas the enediyne antibiotics (e.g., calicheamicin, especiallycalicheamicin gammall and calicheamicin omegall; L-asparaginase;anthracenedione substituted urea; methyl hydrazine derivatives;dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitiaerine; pentostatin; phenamet; pirarubicin; losoxantione;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,22″-trichlorotiiethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (docetaxel) (Rhone-Poulenc Rorer, Antony, France);chloranbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine;methotrexate; platinum coordination complexes such as cisplatin,oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16);ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine;novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda;ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid;capecitabine; leucovorin (LV); irenotecan; adrenocortical suppressant;adrenocorticosteroids; progestins; estrogens; androgens;gonadotropin-releasing hormone analogs; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as antiestrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen(including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON-toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASL® exemestane, formestanie, fadrozole, RIVISOR®vorozole, FEMARA® letrozole, and ARTMIDEX® anastrozole; andantiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF-A expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rJL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELLX® rmRH; antibodies such as trastuzumab and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

Suitable antibiotics include aminoglycosides (e.g., gentamicin),beta-lactams (e.g., penicillins and cephalosporins), quinolones (e.g.,ciprofloxacin), and novobiocin. Generally, the antibiotic isadministered in a bactericidal, antiviral and/or antifungal amount. In acertain embodiment, a compound disclosed herein can be combined with oneor more antibiotics, including, but not limited to, amoxicillin,ampicillin, arsphenamine, azithromycin, aztreonam, azlocillin,bacitracin, carbenicillin, cefaclor, cefadroxil, cefamandole, cefazolin,cephalexin, cefdinir, cefditorin, cefepime, cefixime, cefoperazone,cefotaxime, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, cefuroxime, chloramphenicol, cilastin,ciprofloxacin, clarithromycin, clindamycin, clofazimine, cloxacillin,colistin, dalfopristan, demeclocycline, dicloxacillin, dirithromycin,doxycycline, erythromycin, enafloxacin, enviomycin, ertepenem,ethambutol, flucloxacillin, fosfomycin, furazolidone, gatifloxacin,geldanamycin, gentamicin, herbimicin, imipenem, linezolid, lomefloxacin,loracarbef, mafenide, moxifloxacin, meropenem, metronidazole,mezlocillin, minocycline, mupirozin, nafcillin, neomycin, netilmicin,nitrofurantoin, norfloxacin, oxytetracycline, penicillin, piperacillin,platensimycin, polymixin B, prochlorperazine, prontocil, quinupristine,rifabutin, roxithromycin, spectinomycin, sulfacetamide, sulfamethizole,sulfamethoxazole, teicoplanin, telithromycin, tetracycline,thioacetazone, thioridazine, ticarcillin, tobramycin, trimethoprim,troleandomycin, trovafloxacin, and vancomycin.

In yet a further embodiment, a compound provided herein can be combinedwith one or more steroidal drugs known in the art, including, but notlimited to, aldosterone, beclometasone, betamethasone,deoxycorticosterone acetate, fludrocortisone acetate, hydrocortisone(cortisol), prednisolone, prednisone, methylprenisolone, dexamethasone,and triamcinolone.

In certain embodiments, a compound disclosed herein can be combined withone or more anti-fungal agents, including, but not limited to,amorolfine, amphotericin B, anidulafungin, bifonazole, butenafine,butoconazole, caspofungin, ciclopirox, clotrimazole, econazole,fenticonazole, filipin, fluconazole, isoconazole, itraconazole,ketoconazole, micafungin, miconazole, naftifine, natamycin, nystatin,oxyconazole, ravuconazole, posaconazole, rimocidin, sertaconazole,sulconazole, terbinafine, terconazole, tioconazole, and voriconazole.

The compounds disclosed herein can also be administered in combination,preferably sequentially, with other classes of compounds, including, butnot limited to, antipruritics; anticoagulants, such as bivalirudin;thrombolytics, such as streptokinase; non-steroidal anti-inflammatoryagents, such as aspirin; antiplatelet agents, such as clopidogrel;norepinephrine reuptake inhibitors (NRIs) such as atomoxetine; dopaminereuptake inhibitors (DARIs), such as methylphenidate;serotonin-norepinephrine reuptake inhibitors (SNRIs), such asmilnacipran; sedatives, such as diazepham; norepinephrine-dopaminereuptake inhibitor (NDRIs), such as bupropion;serotonin-norepinephrine-dopamine-reuptake-inhibitors (SNDRIs), such asvenlafaxine; monoamine oxidase inhibitors, such as selegiline;hypothalamic phospholipids; endothelin converting enzyme (ECE)inhibitors, such as phosphoramidon; opioids, such as tramadol;thromboxane receptor antagonists, such as ifetroban; potassium channelopeners; thrombin inhibitors, such as hirudin; growth factor inhibitors,such as modulators of PDGF activity; platelet activating factor (PAF)antagonists; anti-platelet agents, such as GPIIb/IIIa blockers (e.g.,abdximab, eptifibatide, and tirofiban), P2Y(AC) antagonists (e.g.,clopidogrel, ticlopidine and CS-747), and aspirin; anti-coagulants, suchas warfarin; low molecular weight heparins, such as enoxaparin; FactorVIa Inhibitors and Factor Xa Inhibitors; renin inhibitors; neutralendopeptidase (NEP) inhibitors; vasopepsidase inhibitors (dual NEP-ACEinhibitors), such as omapatrilat and gemopatrilat; HMG CoA reductaseinhibitors, such as pravastatin, lovastatin, atorvastatin, simvastatin,NK-104 (a.k.a. itavastatin, nisvastatin, or nisbastatin), and ZD-4522(also known as rosuvastatin, or atavastatin or visastatin); squalenesynthetase inhibitors; fibrates; bile acid sequestrants, such asquestran; niacin; anti-atherosclerotic agents, such as ACAT inhibitors;MTP Inhibitors; calcium channel blockers, such as amlodipine besylate;potassium channel activators; alpha-adrenergic agents; diuretics, suchas chlorothlazide, hydrochiorothiazide, flumethiazide,hydroflumethiazide, bendroflumethiazide, methylchlorothiazide,trichioromethiazide, polythiazide, benzothlazide, ethacrynic acid,tricrynafen, chlorthalidone, furosenilde, musolimine, bumetanide,triamterene, amiloride, and spironolactone; thrombolytic agents, such astissue plasminogen activator (tPA), recombinant tPA, streptokinase,urokinase, prourokinase, and anisoylated plasminogen streptokinaseactivator complex (APSAC); anti-diabetic agents, such as biguanides(e.g. metformin), glucosidase inhibitors (e.g., acarbose), insulins,meglitinides (e.g., repaglinide), sulfonylureas (e.g., glimepiride,glyburide, and glipizide), thiozolidinediones (e.g. troglitazone,rosiglitazone and pioglitazone), and PPAR-gamma agonists;mineralocorticoid receptor antagonists, such as spironolactone andeplerenone; growth hormone secretagogues; aP2 inhibitors;phosphodiesterase inhibitors, such as PDE III inhibitors (e.g.,cilostazol) and PDE V inhibitors (e.g., sildenafil, tadalafil,vardenafil); protein tyrosine kinase inhibitors; anti-inflammatories;anti-proliferatives, such as methotrexate, FK506 (tacrolimus, Prograf),mycophenolate mofetil; chemotherapeutic agents; immunosuppressants;anticancer agents and cytotoxic agents (e.g., alkylating agents, such asnitrogen mustards, alkyl sulfonates, nitrosoureas, ethylenimines, andtriazenes); anti-metabolites, such as folate antagonists, purineanalogues, and pyrridine analogues; antibiotics, such as anthracyclines,bleomycins, mitomycin, dactinomycin, and plicamycin; enzymes, such asL-asparaginase; farnesyl-protein transferase inhibitors; hormonalagents, such as glucocorticoids (e.g., cortisone),estrogens/antiestrogens, androgens/antiandrogens, progestins, andluteinizing hormone-releasing hormone anatagonists, and octreotideacetate; microtubule-disruptor agents, such as ecteinascidins;microtubule-stablizing agents, such as pacitaxel, docetaxel, andepothilones A-F; plant-derived products, such as vinca alkaloids,epipodophyllotoxins, and taxanes; and topoisomerase inhibitors;prenyl-protein transferase inhibitors; and cyclosporins; cytotoxicdrugs, such as azathiprine and cyclophosphamide; TNF-alpha inhibitors,such as tenidap; anti-TNF antibodies or soluble TNF receptor, such asetanercept, rapamycin, and leflunimide; and cyclooxygenase-2 (COX-2)inhibitors, such as celecoxib and rofecoxib; and miscellaneous agentssuch as, hydroxyurea, procarbazine, mitotane, hexamethylmelamine, goldcompounds, platinum coordination complexes, such as cisplatin,satraplatin, and carboplatin.

The disclosure provides a method for inhibiting a cancer and/orneoplastic disorders by contacting or administering a therapeuticallyeffective amount of a compound disclosed herein, derivative or analogthereof either alone or in combination with other anticancer agents to asubject who has, or is at risk of having, such a disorder. The term“inhibiting” means preventing or ameliorating a sign or symptoms of asyndrome, disorder, and/or disease (e.g., tomor growth, cancer cellproliferation and/or migration, cancer cell metastasis, and the like).

The disclosure also provides a method for inhibiting the growth of atumor or cancer by contacting the tumor cells, cancer cells orneoplastic cells with a compound, derivative or analog thereof,including pharmaceutical salt and prodrug forms, with an inhibitingeffective amount. The term “contacting” refers to exposing the cells(e.g., tumor, cancer or neoplastic cell) to an agent. Contacting of anorganism with a topical probiotic composition of the disclosure canoccur in vitro, for example, by adding the topical probiotic compositionto a bacterial culture to test for susceptibility of the bacteria.Alternatively, contacting can occur in vivo, for example by contactingthe topical probiotic composition with a subject afflicted with abacterial infection, a subject susceptible to infection or a subjectsuffering from or at risk of developing a cancer.

Contacting can occur in vivo, for example, by administering thecompound, derivative or analog thereof, including pharmaceutical saltand prodrug forms, to a subject afflicted with an infection, a tumor,cancer or neoplasm. In vivo contacting includes both parenteral as wellas topical. “Inhibiting” or “inhibiting effective amount” refers to theamount of agent that is sufficient to cause, for example, tumor, canceror neoplastic cell death, inhibition of growth and/or migration and/orinhibition of prevention of metastasis.

For use in the therapeutic applications described herein, kits andarticles of manufacture are also described herein. Such kits cancomprise a carrier, package, or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin a method described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers can be formedfrom a variety of materials such as glass or plastic.

For example, the container(s) can comprise one or more compoundsdescribed herein, optionally in a composition or in combination withanother agent as disclosed herein. The container(s) optionally have asterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). Such kits optionally comprise a compound disclosedherein with an identifying description or label or instructions relatingto its use in the methods described herein.

A kit will typically comprise one or more additional containers, eachwith one or more of various materials (such as reagents, optionally inconcentrated form, and/or devices) desirable from a commercial and userstandpoint for use of a compound described herein. Non-limiting examplesof such materials include, but are not limited to, buffers, diluents,filters, needles, syringes; carrier, package, container, vial and/ortube labels listing contents and/or instructions for use, and packageinserts with instructions for use. A set of instructions will alsotypically be included.

A label can be on or associated with the container. A label can be on acontainer when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself; a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Alabel can be used to indicate that the contents are to be used for aspecific therapeutic application. The label can also indicate directionsfor use of the contents, such as in the methods described herein. Theseother therapeutic agents may be used, for example, in the amountsindicated in the Physicians' Desk Reference (PDR) or as otherwisedetermined by one of ordinary skill in the art.

The disclosure also provides method of identifying commensal bacteriathat can produce 6-HAP comprising determining the presence of orexpression of one or more of the sequences of Table 2 (i.e., SEQ ID Nos:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, and/or 55) or sequences that are atleast 98% identical thereto. Method of determining identity and homologyare incredibly well known in the art and have been performed for atleast the past 20 years. Detecing expression of a gene can be determinedby quantitative RT-PCR, southern blot, norther blot etc. Microorganismsthat have an expressin profile similar to S. epidermidis strains M034and/or M038 and containing or expression sequence of SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, and/or 55 can be used in the methods andcompositions of the disclosure.

The disclosure also provide a diagnostic to determine risk or presenceof skin cancer. The method comprising obtaining a microbiome sample fromthe skin of a subject at risk of or having skin cancer and measuring theproduction of a compound of formula I or II or identifying bacteria inthe microbiome that produce a compound of of formula I or II, whereinthe presence of a compound of formula I or II or a bacteria thatproduces a compound of formula I or II is indicative of a cancer or riskof developing cancer. In one embodiment, the subject is a human subject.In another embodiment, the method comprises identifying the presence ofa S. epidermidis strain. In another embodiment, the S. epidermidisstrain has the phenotype of ATCC Number ______ (strain designation S.epi-MO38 UCSD 20180315) and/or ATCC Number ______ (strain designation S.epi-M034 UCSD 20180315).

The invention is further illustrated by the following examples:

EXAMPLES

Bacteria.

Clinical strains of S. epidermidis were isolated from the skin surfaceof healthy donors who had no contact with hospitals over 6 months. Allstrains were characterized by coagulase and catalase activities onrabbit plasma. S. epidermidis strains were further characterized usingAPI-Staph (BIOMÉRIEUX Inc., Lyon, France) and by full-length 16SrRNAgene sequence. S. epidermidis (ATCC12228 and ATCC1457), S. aureus(ATCC35556), S. hominis (ATCC27844) E. coli (ATCC25922) and P.aeruginosa (ATCC14213) were obtained from American Type CultureCollection (Manassas, Va.). GAS (NZ131), GBS (DK23) and MRSA (USA300 andSanger252) were generously gifted.

Characterization and purification of 6-HAP.

S. epidermidis M034 strain was cultured in tryptic soy broth (TSB) at37° C. for 24 hrs and was removed from culture supernatant with 0.22 μmfilter. The filtrated culture supernatant was lyophilized and theresidue was suspended in methanol to precipitate proteins. Thesupernatant was dried under vacuum and the residual substance wasdissolved in water. As 6-HAP is weakly retained C18 reverse phasecolumn, the solution was applied on Sep-Pak cartridge (Waters Co.,Milford, Mass.) and washed with H2O, and eluted with 5% acetonitrile inH2O. The elution was lyophilized and suspended in 90% acetonitrile/10%water. The supernatant was separated by HPLC. After each purificationstep, activity was determined by radial diffusion assay against GAS(NZ131). Purified compound was characterized by Mass Spectrometory andNMR. The purified 6-HAP was lyophilized and dry weight was measured tomeasure specific activity.

Antimicrobial assays.

Radial diffusion assay was performed using GAS (NZ131) strain. Briefly,melted Todd-Hewitt broth (THB) agar (10 mL) was mixed with GAS [1×10⁶colony forming unit (CFU)] and poured in a 10 cm petri dish. Two to fourμL of test samples was applied in a small well punched on the agarplate. Plates were incubated at 37° C. overnight to allow visible growthof bacteria. Antibacterial activity was indicated by the clear zone (nobacterial growth) around the well.

To determine MBC, bacteria were cultured in TSB (Staphylococcus), THB(streptococcus) or Nutrient broth (P. aeruginosa and E. coli) byreaching exponential phase (OD₆₀₀=0.5-0.8). MBC of 6-HAP was determinedby incubating 1×10⁵ CFU/mL bacteria with 2-fold serial dilutions ofsynthetic 6-HAP in half strength Muller-Hinton broth (MHB) in PBS at 37°C. for 24 hrs. After incubation, the number of viable bacteria wasmeasured by counting CFU after spreading 10-fold serial dilutions ofbacteria on suitable agar plates. MBC was determined as a 3-logreduction (99.9%) of viable bacteria after 24 hour incubation.

BrdU Incorporation Assay.

GAS (NZ131) or S. epidermidis (ATCC12228) were cultured in THB or TSB,respectively, by reaching exponential phase. The bacteria (1×10⁶ CFU)were incubated in 1004 of THB or TSB containing 10 μM BrdU, and 25 μg/mL6-HAP or 5 μg/mL mitomycin C at 37° C. until 60 min. After incubation,bacteria cells were immediately fixed FixDenat solution (Roche,Mannheim, Germany). BrdU incorporation into nascent DNA was measured ina time-dependent manner using BrdU incorporation assay kit (Roche)according to the manual.

In Vitro DNA Polymerization Assay.

To examine if 6-HAP disrupts adenosine-thymidine base pair matching inDNA extension, IRDye800-labeled 18-bp primer and 25-bp template whichrequired adenosine (X=T) or cytidine (X=G) at the initial base ofoverhang for extension were designed (FIG. 3e ). The reaction mixturecontained 100 nM primer/template, 0.1 U Klenow fragment (exo⁻) DNApolymerase (Promega, Madison, Wis.), 1 μM dNPTs in DNA polymerasebuffer. The mixture was incubated at 37° C. for 10 min. The reaction wasterminated by adding stop solution (98% formaldehyde and 20 mM EDTA).The extended DNA was separated from primer by electrophoresis on a 20%denaturing polyacrylamide gel containing 7 M urea. Fluorescence wasvisualized with Oddyssey Imaging System (LI-COR Biosciences, Lincoln,Nebr.).

Cell Culture and Cell Proliferation Assay.

B16F10 mouse melanoma, Pam212, L5178 and YAC-1 mouse melanoma cell lineswere obtained from American Type Culture Collection. Pam212, L5178 andYAC-1 cell lines were maintained in RPMI-1640 supplemented with sodiumpyruvate (1 mM), nonessential amino acids (0.1 mM), penicillin (100unit/mL), streptomycin (100 μg/mL) and 10% heat-inactivated fetal bovineserum (FBS) or horse serum at 37° C. under atmosphere of 5% (v/v) CO2 inair. B16F10 cell line was maintained in DMEM supplemented withpenicillin (100 unit/mL), streptomycin (100 μg/mL) and 10%heat-inactivated FBS. NHEKs were obtained from Invitrogen (Lifetechnologies, Grand Island, N.Y.) and maintained in EpiLife medium (Lifetechnologies) supplemented with 60 μM Calcium, epidermal growth factors,penicillin and streptomycin. After 4-hr (tumor cell lines) or 24-hr(NHEK) incubation with 6-HAP, proliferative activity of cells wascolorimetrically determined by monitoring BrdU incorporation with CellProliferation kit according to the manual (Roche).

Gene Silencing of mARCs with siRNA.

NHEK was cultured in EpiLife® media containing pre-designed siRNA formARC1 or mARC2 (Thermo Fisher, Waltham, Mass.), and RNAiMAX® reagent for24 hrs. Cells were maintained in EpiLife for 72 hrs, and incubated with6-HAP (10 μg/mL) for 24 hrs. Anti-proliferative activity of 6-HAP wasdetermined by measuring BrdU incorporation as described above.

HPLC.

The active fraction from SepPak cartridge was separated by HPLC in ahydrophilic interaction mode with Venusil XBP NH2 (5 μm, 100 Å, 10×250mm) (Agela Technologies, Wilmington, Del.) with a linier gradient of H₂Ofrom 5% to 35% in acetonitrile at 4 mL/min. The active fraction wasfractionated, lyophilized and dissolved in 90% acetonitrile in H2O. Theactive fraction was further cleaned with PolyHYDROXYETHYL A (5 μm, 60 Å,9.4×250 mm) with a linier gradient of H₂O from 5% to 35% in acetonitrileat 3 ml/min. Elution profile was monitored with absorbance at 270 nm.After each purification step, activity was determined by radialdiffusion assay against GAS (NZ131). The purified 6-HAP was lyophilizedand dry weight was measured to measure specific activity.

Mass Spectrometry.

A Thermo Finnigan MAT900XL mass spectrometer (Thermo Scientific,Waltham, Mass.) was employed for both low resolution electron impactmass spectrometry (LR-EI-MS) analysis and high resolution electrosprayionization mass spectrometry (HR-EI-MS) using direct insertion probe forsample introduction. The electron energy was set at 70 eV with anemission current of 1.0 mA. High resolution electrospray ionization MS(HR-ESI-MS) analysis was performed on a Thermo LTQ Orbitrap XL massspectrometer. The source voltage was set at 4500 V with a heatedcapillary temperature of 250° C. and a sheath gas flow rate of 60 units.

¹H-NMR.

¹H NMR spectra of 6-HAP were recorded on a Mercury Plus 500 MHz Varianinstrument. Chemical shifts (δ) are quoted in parts per million (ppm)referenced to the appropriate residual solvent peak (DMSO-d6 or D2O),with abbreviations s and br s denoting singlet and broad singlet. The 1HNMR spectrum of 6-HAP in AcOD-D2O (1:5 v/v) displayed two proton signalsin the aromatic region, whereas six signals in DMSO-d6. 1H NMR (500 MHz,AcOD-D2O) δ 8.19 (s, 1H), 8.17 (s, 1H). 1H NMR (500 MHz, DMSO-d6) δ12.74 (br s, 1H), 10.87 (br s, 0.7H), 9.50 (br s, 1H), 8.08 (br s, 1H),7.75 (br s, 1H), 7.48 (br s, 0.4H).

Synthesis of 6-HAP.

6-HAP was prepared according to the previous reported procedure withslight modifications (Preparation of nucleobases and nucleosides asantiparasitic agents, Loakes, D.; Too, K., PCT Int. Apl., 2007135380, 29Nov. 2007). Hydroxylamine hydrochloride (1.20 g, 17.3 mmole) wasdissolved in 20 mL of boiling absolute ethanol and a solution ofpotassium hydroxide (1.12 g, 20.0 mmole) in 4 mL of hot absolute ethanolwas added. The precipitated KCl was filtered and washed three times with2 mL of hot ethanol. Then, 6-chloropurine (300 mg, 1.94 mmole) (Sigma,St. Louis, Mo.), dissolved in 7 mL of absolute ethanol, was added to thehydroxylamine solution. The reaction was refluxed for 2 hours thencooled to room temperature and allowed to stand overnight. The whiteprecipitate formed was filtered and washed thoroughly with water andthen ethanol, and dried under high vacuum to provide 6-HAP (230 mg, 78%)as a white solid. ¹H NMR (500 MHz, DMSO-d6) δ 12.84 (br s, 1H), 10.92(br s, 0.5H), 9.48 (br s, 1H), 8.08 (br s, 0.6H), 7.77 (s, 1H), 7.48 (brs, 0.7), in agreement with those reported. The generated 6-HAP waspurified by HPLC using Venusil XBP NH2 and PolyHYDROXYETHYL A asdescribed above.

Animals.

All experiments involving animal work were in accordance with theapproval of the Institutional Animal Care and Use Guidelines of theUniversity of California San Diego (Protocol number: S09074).

In Vivo Skin Infection Assay.

Dorsal skin of C57BL6 mouse (6-8 weeks female) was shaved, treated withdepilatory cream and rinsed with water at least 24 hrs before bacteriaapplication. The shaved skin was cleaned with alcohol swab twice toremove originally colonized bacteria. S. epidermidis (M034 or ATCC1457)were cultured in TSB overnight, washed with PBS, and then re-suspendedin PBS. GAS (NZ131) and MRSA (Sanger252) were cultured in THB or TSB,respectively, until exponential phase (OD600=0.5-0.8), washed andre-suspended in PBS. Mouse dorsal skin (2×2 cm) was applied with eitherstrain of S. epidermidis (1×10⁸ CFU/10 μL) or PBS (10 μL) for 2 hrs. GASor MRSA (1×10⁵ CFU/104) were epicutaneously challenged on the dorsalskin for 6 hrs. Live bacteria were harvested with a Catch-All Swab(Epicentre Biotechnologies, Madison, Wis.) pre-wetted with TSB or THBfrom the skin surface (2×2 cm). Bacteria were suspended by vortex swabhead vigorously in 1 mL THB or TSB. Ten-fold serial dilution of thebacteria suspension was spread on a blood agar plate or mannitol saltagar plate to count CFU. GAS (hemolytic) were distinguished from S.epidermidis (non-hemolytic) on a blood agar plate and MRSA (mannitolpositive: a large yellow colony) were distinguished from S. epidermidis(mannitol negative: a small pink colony) on a mannitol salt agar plate.

For subcutaneous infection, mouse dorsal skin was subcutaneouslyinjected with GAS (1×10⁷ CFU/50 μL in PBS) by 31G needle. The mice wereintravenously injected with 6-HAP at a dose of 20 mg/kg weight or equalvolume of vehicle (2.5% DMSO in 0.9% NaCl) after GAS infection. Wounddevelopment was monitored everyday by taking photographs along with awound ruler and lesion size was measured with ImageJ([http://]rsbweb.nih.gov/ij/)(hyperlink disabled by brackets).

In Vivo Tumor Growth Assay.

B16F10 were suspended in sterile PBS. Shaved mouse dorsal skin wasintradermally injected with 3×10⁵ cells/50 μL. The C57BL6 mice weresubsequently injected with 6-HAP dissolved in 2.5% DMSO/0.9% NaClsolution (40 mg/mL) at the dose of 20 mg/kg mouse via intravascularroute every 48 hrs for 2 weeks. Control mice received an equal volume ofvehicle. Tumor size was measured as the two perpendicular diameters witha caliper and volume was estimated by a formula, width²×length/2. Themice were sacrificed when tumor size reached >2 cm according to theanimal protocol.

UV-Induced Tumor Formation in SKH-1 Mice.

Female SKH-1 hairless mice (4-week old) were purchased from CharlesRiver Laboratories (Wilmington, Mass.). The back skin of mouse wastopically treated with a single application of DMBA (200 nmoles/100 μLacetone) as a tumor initiator. A week after tumor initiation mice wereirradiated with 180 mJ/cm² of UV-B twice a week, and were simultaneouslytreated with epicutaneous application with live S. epidermidis MO34 orATCC1457 strain (1×10⁷ CFU) 6 times a week for 12 weeks. Tumor incidenceand tumor number in each mouse were recoded every week.

Statistic Analysis.

Statistical analyses were performed using GraphPad Prism 5 software(GraphPad, La Jolla, Calif.). Independent t-test was used. Independenttwo-tailed t-tests were used to compare experimental and control groupsfor significance of differences (P<0.05).

S. epidermidis Strains from Human Skin Produce Non-ProteinaceousAntibiotic.

To screen for commensal skin bacteria producing antimicrobial activity,44 strains of S. epidermidis from clinical isolates of normal human skinwere cultured overnight and the antimicrobial activity produced by eachisolate was determined by radial diffusion assay of their conditionedmedia against GAS (FIG. 1a ). Thirty three strains produced detectablezone of GAS growth inhibition. The S. epidermidis ATCC1457 strain didnot show activity and was used as a negative control. Some specificstrains, named as M034 and M038, produced the largest zone of GAS growthinhibition and were selected first to characterize the most potentantimicrobial molecules.

To characterize the molecule(s) responsible for antimicrobial activitysecreted from the M034 strain, the antimicrobial molecule was purifiedfrom the conditioned media based on activity against GAS. A single peakwas isolated in the last chromatography of 5 purification steps (FIG. 1b). The final yield of purified compound was 7 mg from 6.4 L culturesupernatant. This purified fraction showed strong zones of inhibition ofGAS growth, and was found only in the culture supernatant of M034 andM038 strains, but not in laboratory strains such as ATCC12228 andATCC1457 strains (FIG. 9). This antimicrobial molecule was heat-stable(FIG. 1c ) and protease-insensitive (FIG. 1d ), thus suggesting theactivity may not be a protein.

Skin Colonization by Antimicrobial S. epidermidis Strains ProtectsAgainst Pathogens.

To examine the physiological relevance of colonization by the strains ofS. epidermidis that produce non-proteinaceous antibiotic on the skinsurface, M034 or a control strain of S. epidermidis without detectableantimicrobial activity (ATCC1457) (FIG. 1a ), or the vehicle alone, wasapplied to the surface of mouse dorsal skin. 2 hrs after applicationthis site was then challenged with defined amounts of GAS or MRSA. Skincolonized by the antimicrobial strain inhibited growth of the pathogenswithin 6 hrs, but skin colonized by the control strain or vehicle didnot (FIGS. 2a and b ). These data suggest that colonization by thisantimicrobial S. epidermidis strains is protective against microbialpathogens on the skin surface.

Structural Analyses of Antibiotic Produced by S. epidermidis.

High-resolution electrospray (ES)-mass spectrometry analysis of thepurified antimicrobial fraction identified a molecule with a mass of151.0487, predicting a molecular formula of C₅H₅N₅O (calculated:151.0489) (FIG. 3a ). When S. epidermidis was cultured in the presenceof ammonium-¹⁵N chloride, the isotope was incorporated into the nitrogenatoms of this molecule, indicating that it was produced via de novosynthesis, but not by fermentation or breakdown of components in theculture media (FIG. 3b ). The ¹H NMR spectrum of the purified compounddisplayed two proton signals in the aromatic region (M=8.19, 8.17),whereas six signals in DMSO-d5 (8H=12.74, 1H; 10.87 0.7H; 9.50, 1H;8.08, 1H; 7.75, 1H; 7.48, 0.4H) (FIG. 1c ). The gHMBC spectrum ofpurified compound revealed five carbon signals in the aromatic region(6C=113.60, 144.94, 148.17, 150.28, 150.45) (FIG. 10). These NMRchemical shifts suggested the presence of a purine moiety with anadditional oxygen atom attached to one of the five nitrogen atoms. Giventhe chemical formula as C₅H₅N₅O, the structure was predicted as6-N-hydroxyaminopurine (6-HAP). To confirm this predicted structure weperformed a chemical synthesis of 6-HAP. The natural compound had anidentical chemical shift to synthetic 6-HAP by ¹H-NMR (FIG. 3d ). Inaddition, the fragmentation profile of natural compound byelectron-impact MS (FIG. 3e ) also matched that of synthetic product(FIG. 30. Most importantly, the antimicrobial activity of synthesized6-HAP against GAS was comparable to that of natural product (FIG. 11).Thus, the combined data indicated that antimicrobial activity producedby this S. epidermidis strain is mediated by 6-HAP (FIG. 3g ).

Antimicrobial Activity of 6-HAP In Vitro.

To examine the specificity of 6-HAP for various bacteria, minimalbactericidal concentrations (MBCs) were determined according todose-dependent killing curves at 24 hrs of incubation in vitro (Table1). 6-HAP killed or suppressed growth of several major skin pathogensincluding GAS (NZ131), group B streptococcus (GBS) (DK23),methicillin-sensitive S. aureus (ATCC35556), methicillin-resistant S.aureus (MRSA) strains (USA300 and Sanger252), Pseudomonas aeruginosa (P.aeruginosa) (ATCC14213) in vitro. In contrast, 6-HAP showed weakantimicrobial activity against Staphylococcus hominis (S. hominis)(ATCC27844) and Escherichia coli (E. coli) (ATCC25922) andPropionibacterium acnes (ATCC6919). S. epidermidis (ATCC12228) was alsoresistant to 6-HAP.

TABLE 1 MBCs of 6-HAP against indicated bacterial strains. BacteriaStrain MBC (μg/mL)* GAS NZ131 0.156 GBS DK23 0.781 S. aureus ATCC355566.25 MRSA USA300 6.25 MRSA Sanger252 0.781 E. coli ATCC25922 50 P.aeruginosa ATCC14213 1.56 S. hominis ATCC27844 12.5 S. epidermidisATCC12228 >50 P. acnes ATCC6919 >100 M. luteus ATCC4698 12.5 *MBCs weredetermined as a 3-log reduction of viable bacteria after 24 hourincubation at 37° C. in half strength of MHB in PBS. After incubation,live bacteria were measured by counting CFU after plating serialdilutions on agar plates. The data are representative of threeindependent experiments.

Antimetabolite Mechanism of Action of 6-HAP.

As 6-HAP is structurally similar to adenine, we hypothesized that itinhibited bacterial growth by a different mechanism than membranepermeation used by most other skin surface AMPs produced by the hose, orbacteriocins previously described from S. epidermidis ^(17,18). Toexamine this we first compared the kinetics of killing by 6-HAP to thoseby LL37, a potent human AMP with capacity to disrupt membranes, andmitomycin C, a known DNA synthesis inhibitor. Growth of GAS over timewas inhibited by 6-HAP at a rate similar to mitomycin C but slower thanLL37 (FIG. 4a ). LL37 disrupted permeability of the GAS membrane after1-hr incubation, whereas 6-HAP and mitomycin C did not (FIG. 4b ). 6-HAPalso did not directly affect plasma membrane permeability of normalhuman epidermal keratinocytes (NHEKs) or the human sebocyte line SZ95after 6-hr incubation (FIG. 12).

We next assessed the effect of 6-HAP on DNA synthesis. Significantsuppression of BrdU incorporation into the genomic DNA of GAS could beobserved in a time-dependent manner (FIG. 4c ). However, 6-HAP did notaffect BrdU incorporation in S. epidermidis (FIG. 4d ). To directlyexamine the action of 6-HAP on DNA extension in a cell free system,25-bp templates and matching 18-bp fluorescence primers were designed tomeasure DNA extension in vitro by Klenow (exo⁻) DNA polymerase (FIG. 4e). In the presence of 6-HAP, we observed synthesis of the expectedextended DNA product when the template required cytosine (X=G), but lackof extension when adenosine was required (X=T) (FIG. 40. These datasuggest that 6-HAP inhibits DNA synthesis by interfering withadenosine-thymidine base pairing. Indeed, addition of excessive adeninepartially decreased antimicrobial activity of 6-HAP against GAS (FIG. 4g).

Mechanism for Selective Antiproliferative Activity of 6-HAP.

Given the capacity to inhibit DNA synthesis, we next explored theanti-proliferative effects of 6-HAP on mammalian cells. 6-HAP inhibitedBrdU incorporation in several tumor cell lines, including Pam212squamous cell carcinoma (FIG. 5a ), B16F10 melanoma and L5178 and YAC-1lymphoma (FIG. 13). In contrast, BrdU incorporation in normal humankeratinocytes (NHEK) was not affected by 6-HAP until a very high dose(100 μg/mL) (FIG. 5b ). The mechanism of selective inhibition ofmammalian cell lines, as well as selective inhibition of bacterialpathogens, is currently unknown and a subject of ongoing investigation.

Because some nucleobase analogs exert mutagenic activities due to theirmisrecognition of wrong bases, mutagenic activity of 6-HAP wasdetermined by detecting mutagenic events at the thymidine kinase (tk)locus of L5178Y tk^(+/−) mouse lymphoma cells. This sensitive assay ofmutagenic activity did not detect a difference between 6-HAP compared tovehicle, whereas treatment with methyl methanesulfonate was a positivecontrol for the assay and did induce a high mutation frequency. Thesedata are consistent with a lack of association between S. epidermidisand neoplastic transformation despite the chronic presence of thismolecule as a product of the normal commensal microbiome.

In mammalian cells, mitochomdrial monoxide reducing components (mARC) 1and 2 have shown to be capable of reducing N-hydroxylated nucleobaseanalogs to canonical nucleobases. Relative expression level of mARC1 andmARC2 was much higher in NHEKs than that in cancer cell lines, such asPam212, L5178 and B16F10 (FIG. 5c ). Thus, we hypothesized that mARCsmay contribute to detoxification of 6-HAP in NHEKs. Gene silencing withsiRNA significantly decreased expression of mARC1 and mARC2 in NHEKs(FIG. 5d ) and increased cellular sensitivity to 6-HAP, suggesting thatmARC2 protects cells from 6-HAP.

Effect of Systemic Administration with 6-HAP on Skin Infection andMelanoma Growth in Mice.

As 6-HAP exhibited both antimicrobial activity and antiproliferativeactivity in vitro, we next explored the systemic activities of thiscompound in vivo. Repeated intravenous injections of mice with 6-HAP ata dose of 20 mg/kg every 48 hrs for 2 weeks resulted in no apparenttoxic effects as assessed by visual appearance, behavior or change inweight (FIG. 14). This response was consistent with prior results inseen in FIG. 4i demonstrating a lack of inhibition of the proliferationof rapidly dividing normal keratinocytes. Thus, given the apparent lackof toxicity, the effect on deep tissue infection was tested byintravenously injecting the non-toxic dose of 6-HAP (20 mg/kg) into amouse model of GAS deep skin infection. A single injection of 6-HAPafter inoculation of GAS significantly suppressed the clinical lesionsize (FIG. 6a-b ) and GAS survival (FIG. 6c ) in mice.

Similar to our experiments to evaluate the antibiotic action of 6-HAP,we also examined the activity of 6-HAP to inhibit tumor growth in vivo.Mice were intradermally inoculated with B16F10 melanoma, followed byintravenous injection of 6-HAP (20 mg/kg) or vehicle every 48 hrs for 2weeks. Tumor size of this aggressively growing tumor was suppressedby >60% in mice receiving 6-HAP compared to those received injectionswith vehicle (FIG. 6d-e ).

Effect of Skin Colonization by S. epidermidis on UV-Induced Skin TumorFormation.

Given the data that 6-HAP inhibited the proliferation of several tumorlines in vitro and in vivo, we hypothesize that colonization by strainsof S. epidermidis producing 6-HAP is protective against skin tumorformation induced by UV-B irradiation. To address this hypothesis,two-stage carcinogenesis model was employed. SKH-1 hairless mice weretreated with DMBA for 1 week, followed by UV-B irradiation twice a weekand epicutaneous application with S. epidermidis 6 times a week. Miceinoculated with S. epidermidis ATCC1457, a non-6-HAP strain, elicited88% tumor incidence at 9 weeks (FIG. 7a ) and multiple tumor formationsby 12 weeks (FIG. 7b-c ). In contrast, repeated application with S.epidermidis M034 strain producing 6-HAP significantly decreased tumorincidence and numbers (FIG. 7a -b, d). Histopathogenic examinationdistinguished that SKH-1 mice colonized by control strain developedsquamous papillomas (FIG. 7e ). The papilloma development wassuccessfully inhibited by epicutaneous application with 6-HAP strain(FIG. 7 b, d, f). These data suggest that 6-HAP produced by S.epidermidis contributes to the resistance to neoplasm formation on theskin surface. In addition, both strains penetrate tumor and dermis ofskin (FIG. 7g-h ), suggesting that the direct interaction between S.epidermidis and tumor cells or skin residing cells. This data isconsistent with our previous observations that a part of skin microbiomecan penetrate the epidermal barrier of human skin.

S. epidermidis Strains Producing 6-HAP in Human Skin.

6-HAP was detected by HPLC of culture supernatant from two distinctclinical isolates of S. epidermidis. To further explore the frequency of6-HAP production in human commensal S. epidermidis stains, whole-genomesequencing of the MO34 strain was performed and used for analysis ofexisting metagenomic data sets of the human skin microbiome. Sequenceanalysis frequently identified S. epidermidis strains similar to the6-HAP-producing isolate within the human skin microbiome but detectedsimilar strains at a different frequency at distinct body sites.

TABLE 2 28 marker genes that are present in 6-HAP-producing S.epidermis strains but not in any of the other strains. Hypotheticalgene ID Role Sequence peg.44# hypothetical SEQ ID NO: 1 proteinatgagtgctgttttattatcagcaattagtccaacggcaagtgtaaatgaatcaatagaattgtacaagtcagttagcaagtcgaatatagaaattgaaaaagataataagactttagatgattcgttttaa peg.45# hypothetical SEQ ID NO: 3 proteinatgattcgttttaaaatcctaaatatggtaactgaacagactattaatagtttaccatataatcttaaagaagggacttcagaaagaattggtggttcctttactgtaaaaaaggattaa peg.131#YefM protein SEQ ID NO: 5 (antitoxin toatgactgttaaatcctattcatatgtacgtgaacatttcaaagacatgattaataaagtta YoeB)atgatgatagcgatacgataacaattacaacaaaagaccgtaatgcagttatgatgtcagaagataattataatgaaataatggagacactatacttgcaacaaaatcctgccaatgcaaaatatttatcagaatccattgaaaacctagaacgtggtaatataaaaactaaggatattttgatataa peg.139# antirepressor SEQ ID NO: 7 [Staphylococcusatgttagataaagaaaagcaaaaagaagctatgcctattattcaaggagagtacaaa epidermidis]tcagatgaaccaattaattacattaaagcaaacacaatcccaaacaaagctacatcaacgacttttggatatgaaaagatgattagtaaagaggctatgacgccagaaatgttagaggttcgtcaaataattcttaatgatgtggtaaagctcacagaatcaatgaatcaatttaaacttgatattaaagttagtaaagtaatttatgacaaatatggggtgagataa peg.140#antirepressor SEQ ID NO: 9 [Staphylococcusatgattaaacaaattttcaatgataaagaaattcggtttatcgaaaaagaagatgaatatepidermidis] tgggcagtagctggagatgtggcaaaggtattggggtactcacatacaccacacatgactagattattagatgaatcagaaaaggctgtccataatgtggtcaccgttaaaggtaaacaaaatgctgtgatcatattagaaataggtatttatgaagctatttggaatagtagaagagatgaagctcaagaattttag peg.141# XRE family SEQ ID NO: 11transcriptionalgtggttaacaacgttaaaagaatacgaaaaaataaaaaaattaccattactgaattaa regulatorgtagaaaaagtgggataagcagaacaactatatacaaattagaatctcaaaaatcaa[Staphylococcusatcccagcttggaaaccattcaaaaaatatcttctggtttagatgaaaaaccagaaaa epidermidis]aatttttaacctcattgttattcaagaattacaaaaggagccttaa peg.142# XRE familySEQ ID NO: 13 transcriptionalgtgaatgactttggaaagaaattaaaagaattaagaggcgaccaatcaattagagaa regulatorgcctctaggaatattggtataagtcacacttatttagatagtttagaaaaaggtattgat[Staphylococcusccaagaactggcaaagaaagaaaacctacaattgaagtaattcataaactatcaaaa epidermidis]tattataatgttgatttttttgatttaagcagattagcaggtgtgtttgtatcaattaaagatacgcctaaagaagataagcgagaagaaattaacaaaatgaagaaaagatttaaagaatattttaacgatacagaacttattgttaaagaaaattatcttgatattatgacaaaaaagttaagttatcatgaaattattttttggcaaaatttatataatttttatattcaagaaaaagattctgattatctaaaaataaaagatgaagaagatacagatattttaacatttatagcttccttgtttaaaatattaactgaaaataaaaattctaatgatgacgaaatatttaaaggcatttcgaatgattttaataaattcttaaaatcatacttaaatattaagtag peg.300# C4-SEQ ID NO: 15 dicarboxylateatgattaacactgccgtcactggtgctgaacctggaggtaactggaatccaaatatcaABC transportertgataagctgcgctactatagcaaagatgccacatcccagtgcaccagcagttctta[Staphylococcusaccctaataatatcgctagaatcatgattatgatttctatgataaacaataacatggaag epidermidisaatctcctaacttatgccgaacaattttcacttcaatattttatcactttaatatgtaa CIM28]peg.349# uncharacterized SEQ ID NO: 17 proteinatgccggaagtttccggagaaatcgcttttaatttttcagcaattttagccgcagctataacaagtgataaagttgatgtaagtaagaaagcagatgcaattgtagttttggtgtcaaaccagcgtctaatgtaa peg.372# MFS transporter SEQ ID NO: 19 [Staphylococcusatgccggcaccaatcgcttggataatccgagatatcattaaaatagaaaaagtaggt epidermidis]gaaactgctgctacaacagatcctataagaaaaattgccattgagaaaatatataaat ga peg.505#hypothetical SEQ ID NO: 21 proteinttgcctaagacacctgaagatgggaaaccaagtgtagataagaaaagctttgtaagtgaaactattttatttttatttatgtcgattctaggagaagtttttggctatgaagatgaaaaagatggacaacttgtacatgatatttctccaataaaaggacaagaaaaaaatatagaaaattctggttcagatgtgtttttctcttatcatgtagaagacgcaatacatccatataaaccagattatcttgctttatattgtttgaaatcggatcatgagaaaatagctataacagagacatcttctattagtgaagcaatgaaaagattaagtacgtcaacgcttaatattcttagaaaacccatgtatgaattacatccacctgcttcttttaattcaagtcatttatctagaaaagtatcagtaataggaggaagtcaaaagaatcctgaattattaattcatgaaacattaatgcaaggaatagaagatgaagcagaaaaagcattaagtgaattgaaagaaacgttaccggaagtttcaaatggagttcaattaaacccaggagaacttattattttagataataataaagcagcacatgctagatcagcttttaaacccagatatgacggtgaagatagatggctacaaagaatgttttctattaatgacttaaaaggattagaggattacatgaaagaagacgaaaatatatttgtacccttggtggatatattaaaagacaaataa peg.742# hypotheticalSEQ ID NO: 23 proteinatgaactttaaaacgaattttaaaggtttgtttagtatagaaaagaaatttaatgtaaatttattgccatctcaactcaaattagaagataaaataaacattttttgggcaacgttaatgagggttgtaggctggcttttatatacattattaattgcaagtatgactgaagttaaaaataataatattattatatacatatcatgtattttgatagtagtcttattaatatttgatataagttcaatctttcttatttctgatgatatgagaaagaaaaattatagatatttgtttatcaaaagagacgaagaatattatcgtttagataaatatttttacgatatttctgataaacaagttttacaatatactgtaaatgaaaaagatatgaaaattgaaaaaaataatggcgatccaaaaattgatgagaaagaaaaaatagttggaaaaaagattgttactgaaaagataaatagtgataaagtcaatacatttatccaaactgatagcgaggctacatatagtagcaaatatattactttagtacctattgttcaaaacattatattgataataagtataatattaacagcttgttttcataaactttggattgttatattatgtcttatcatatattttttgttaaatggaatatttacactacgattatcatctaatgtaaatgaaactttagaaaataacaatataaagcaagttaaaaataaagtgaaaaatcattttaatgaagacgaaaatgataatgttggagataaaaaggatagctatgaacaccgtgataagaatatcaatattatcgttgttaatgaactaaaaaagtaa peg.743#terminase small SEQ ID NO: 25 subunitgtggctacaccagtgtttattgatagtgttggggaagaagatgaaaagaatgagaga[Staphylococcusgatttagaaaagttaagtaaactatatcctaatgcagagtttcacattgatgatattaggepidermidis] tag peg.1065# membrane SEQ ID NO: 27 proteinatggctgttggattaccgcctgcaactgctttaggtacaaataaattagcgagttctttc[Staphylococcusggatctcttacagctactatctcctttattagagcaaaaaaagtaaatattaaattgatgepidermidis]cctaaaatattcccttttgtttttcttgcttctattattggagcttatgttgcaactgtaattccagcgcgatattttaaacctttggcgattataatgctttttatagtattgatttatacactctttaaaaaggattggaacggtaatacactaataaacgaaacttctaaattaaaattatttattgttttttccctactaatattaattggcttctatgatgggtttttaggtggaggaacaggtagtttctttatatttgttttattaattttaggattagactttttaaaatctgcaggaaatgctaaagttttaaattttggaagtaatataggtgccctgcttttatttatattattaaataaagtagattatttattaggtttcagtatggctttgtctatgattgttggaagctatattggtagttcattcgcagtaaaaaaaggtgtttcatatgtaaaattattatttataattgttactttacttttgttaataaaaaacttatatgattatatatttcagtaa peg.1086# hypothetical SEQ ID NO: 29protein atgaaattactagatgacaataattttgacttaaaactacctattaactataaaatcaatactgataattatagatcacttactcaagataaattagatgcattaagttcatctgatgctaattttgatggtatttgggattag peg.1366# LysR family SEQ ID NO: 31transcriptionalatgaatcttttaatcagagaattggaaaatcattcaagagaagttggactcatagcata regulatortaataaagaaatattgcaaaaagtatggagtattttaagtgaatacatagtgtaa [Staphylococcusepidermidis] peg.1369# ABC transporter SEQ ID NO: 33 permeasettgagagatccaagccaaacattattgattaaaaagttaaaaggtatatatttcaaggc[Staphylococcusttttctaacttcatcaccatattctggaattaatggaactaaattagttgctataggtacgepidermidis] attaatataagaaaaaccccaattgaaaatactaaagaagaactttttactaataaagtaattagaaatagaaatagaccataa peg.1452# hypothetical SEQ ID NO: 35 proteinatgaaaaagatgtggttaccaattataactacaatcatagtagctataattatagtattaatcattttaaaaaagacaaatcatttatattttaacaacttggatacatataaagtttataaagtggaagatagaaaagacatttctggtaagggtatcgtttttcctgaacatgttaaagtgtataaaattaataaaaatataggagaatatattagaccacaaattaaagattttagaaaagtgaaaaaaggtactccccttatttattatgatactaatagtagcaacaggcctaatctagtggataatattaataacgttaaagaagatttaaatcgtgattatcaaaacgtagctaaacacaatagcagtagttatcaaaagcaaatatctaatgattaccaaaggttatttaaagctcaacaaaaattaaatcagcatgataatctgtctagtaaagatatatatgcagcttttaatggtgaagtgaaaattagtaattcaattagcggtaaaaatggtgacgaaattttaaaattagtttctacaaagtcagttattaagatgagagcttcacaatatatagttaataagataaaaaaaggaagtaacgttaactttaaattagatagtgacagtgaaaatgtaaaaggtaaggtccagtctattgataacttacctataaatatgaaaaaagaacgttactataaaaactatgaacctaaatatatgattacaatttctgatttaaatcatggtgtgagagcaggatttactgggaaagtaacgattccttataatatgattgaaattcctcaaaatagtatcattaataaaaattatgtttttatagttaataaggataattatgtacaaaaacgtagagttaaaatagtaaagattaaaaataaactcattgctaagaaaggattaaaattaggtgatcgagtcattgaaaaacctaaaagatctttacaagaaggacaacaaattaatattaaataa peg.1474#MULTISPECIES: SEQ ID NO: 37 pathogenicitygtgcaagaaactcattacgaatttaatatagatgatgaattaagaaaactaggtttattaisland proteingttggaatatccgaagaaatgtactactgctcaattagtcgaatatcaacattgtatctt[Staphylococcus]gaaaactttggaactaaatgggtagcatggcgtgaaacttatgattttaagaatgataaaagagtatcgcacagaataatagcaaatggcagttttgaattagtagctgcaagaactaaaaactatttaaactacattaaaagaaagcagggaataaaatga peg.1475# DNA primaseSEQ ID NO: 39 [Staphylococcusatgaaaatgtacaatgcagcaaagaatctccttagtaaagatgtgcaagttgtacccttsaccharolyticus]aaacgataataaaaagccaacagtatcatttaagaatataactattgatgatgattttatagataacaattttttagcatatgcaaagacaaatgtattaggtgtcctaactcgtggtttatggtgtatcgacattgatattaatcatgtaaatggtgaaagtggctttgatagtttgaaagacattccttactatgctgagtttgtttctaatgcacaaaatacgctagtgcagacaacagcaagtgggggaaagcatgtaatatttaaaaaacgtgatggtgttgaatacgctcagaaaataggatatttaccatcagtagatattaaagcacatgataataactattttgtattagctggaagtaaaacagctaaagggctatacactagtaataagaaaccagtaatcacttatgatggtgaatttgaagatcgcattttttcaaaacgtggaaattatctacaacagactatggaaaagttctcagtaaaaagcgtgttgcctaatcacaattttaatcatttacaacatactggcaaaggtggactaggtaaagaggcatacaatcgtgtaattaatggtgaaagcatagaacgtaataatgatgtatataaagctattagttacgctttacaatgtaacgtggatatagagcctctaaaagtaattattggtgatgttaaagcaaatggtgatgaatttactttagaagagtgggaggcttcatataatagtgcaagaaactcattacgaatttaa peg.1476#phage resistance SEQ ID NO: 41 proteinatggacgaagtttctttatataaaaaacattatcaattccattctaaattagataatgttga[Staphylococcustacacctaatttatctcgtataaaagaaattagtaaaagaatttactttgctgcaattacasaccharolyticus]acagaaaaacaaatttttaataataaaggaagtgtttatcaccaaacaaaagatgaatttgcaggtgattacattaataaccttactttagattataccataaaacctagagaaataggtgcagtctatggaactatctctgttaaaacaacagtagagaacggtgaggagaataaagaggcacattttaagcctagtaaaacaaatagctatgcaaagttcattattgatctaattactgaaaaagtcatctactcaaaagagttggatagctttatcaaattaaatagcaatcaatatgaaattatagataatactaatttttcattagagtatccggtagacaataagtatcatattaatgattttcttgatgtaatgctagaagtctacaaagagtatttcattaatgattatcaatataatatttatccttacgctttcgcaggtaatgactggatatataattgcagaaaattagaatttgtagataaaaaaattactagtaacgattactatatcatcaaatatgatgtagataagaaaaatataaacactcaattagcacaaaaattttttgacttagtaagtgacaatgaacgcagtaagaataatttaatgttggtacacgcttatactatgtatcgaaaaatgaaacttattcaagcagaaaaatggttcttaatcaaagactttgggcgatctggtaaaggtttatttatggaaacttttgaaaaacttctaaatgtaaacaaagtcaattttgatagcttattatcatctggctttgaggctgcaaatgaatggcttaacttttatggtgcagatattgctcatgcaaatgaaacaggcgaaattaataaaggtatgatgagaatattacgcaaaatagctactggtgagaatatttcagggcgtggcatacaacgaaataacgttaagtttaaaaataatgcagtattaattttagatactaatgaaagtgttgatactggtgaaattacagcaaatagaacacgtacagttaaaatcgcatttaaggatagaccaaagaatgaaactgatgaagaacgttatcaagtatttaaaccattttgggactttgttaagcctaacgggaaaaactcagtcaatgcgtcagtatcatttttaatattaagtcttgagtatcttaaacaaataggcagagaatttaagttcaataacgtaacacttaaaaactattacaccgaagatgaattgacggacactcaaattcttatgctcaaagtcttagctaaacaagattttattttttcaggtgatgagatactacaaaaaactattgaagaagattataaaaatctgaaatataaaaaagcaaaagaagatatgaaaaaaataggagtggctattaaccaacaggaatggatagagggacaaaacactaaagttcataaagtgaaaaatcaagaattatttaatatggctttagctttgattgaaac ttagpeg.1542# hyperosmolarity SEQ ID NO: 43 resistancettgcttgatgaagttgttattctatatttttcatttaatggttttatatttactgtaaacgaaacprotein Embaatactttcattcccacttttatctgtggctttaacttttacaattttatttgtgctattagttac[Staphylococcus attaggagcctga epidermidis] peg.1635# hypotheticalSEQ ID NO: 45 proteinatgaataaattaaataacaaagattataaaaatattgaaggcaaattgaattacgatcatatcgtaaatggcaaaaagcacattaaaaaaatgagcaaactattacaaaaacgtcgtaacaaagatatttcaattattaaaaaaatgtacccttatttaaataataatgaaattttagaaatcactaatgattatcaagaatacaaaaatcttgttcaagctactgaaacttttacagactttcctagcatttacgaaggttctaatattagtaaattcttaactgaagatgatattgcagatttaaaaatggctgttgaagaaatgctagcttttgttgaaagattggaggagtag peg.1638#hypothetical SEQ ID NO: 47 proteinatgctaaactttgagttaaagaaacacttaaaagataaagatatgactattagtgaattaagcgaaaaaactggaatatcaagaaattctttaggattattaataaatggaaaaagtagaggagttcaatttgaaacacttgaaaaaatttctagagttatgaatgtagatatcaaaaatttattttcaatgacttttgactttatagaaatatctgcaaaaaatgaaaaattacgtagttctgaagtaggagttgattacaatgcgtcttatgattttaaacaattagtctgtaatatgaatatagatggaacagaatatgaattttctgttcaatatgaaatagatttacagttaaacatattaaaagatcatagttctgaagttaaaattactattgatttaagaaagtttaactacctaaatttatttttacccatcaattctgacgtagataatgaaatagcatatttaactcatgtttattttatagacactatactcaaaatcaataaagatgaaattttagaattaatgggtaaaggtataaaaatatcttcaaatcaaatttcttatgtaataataaaagacgcttttcacattatttcatctggtattttatatatgaataatttcaagattacacaaaaagagttttatgttaataaactcaaaactagtaatactataaattacatcgaagactccaataaaatactttttacaagcaggcataaaaatgttacataa peg.1640# hypothetical SEQ ID NO: 49 proteinttgtcgattttaattagttttcttagtttaattttatcaactttcgcttttttatatgcgaacaaaagacataagttgaatatgttagatcatttagatgataaatcagaatggagaaaaaaactatttgaaattgcaggttcttcaaaaattggaatgggaaatttatatcaatttagagctgcattaagattcacatacaaaaatgaagatgaatattatgaatataattattttgagtgcatgaacataattattataaaatattgtgaaaaattaataagtcaaaatcgaatagaagatcacaaacacaacgaaaacgaaaaggatcaatcttatttaaaaaattatgaaatggattcaattagattattttgtatttacatgttagcagatcattgggaaaaaaaacaaaacaaaaattttaaatttaacaatccagtaaaagaagtagaattatgtatagacaccttacaaaaatttctcaatattaatgataaaaactattgttataaatgccataaaagtaaattaaatagagataatttttactgtttgtatcaccaaagtataagtttgataaattctatgacatcctaa peg.1975#ABC transporter SEQ ID NO: 51 ATP-bindingatgggctgtacgactgctgatattttagcctgttttaatcctaatctatatatttttttaagat proteinttgtatgcgcattatctaactctaaacgctcagtattagatactttaactaaacgcatttc[Staphylococcusggtaagcactctacctaataatccactgaagttcgcaatttcagattgcgtattggtag epidermidis]atattttttgcatcacacgtcccagaggaacgatgattaaaataaatacaggaatagtaataaatgttaataaagttaatttccaatccatgataaatagcatgactaatgaacctactaacgttaaaacagaaggcaataaattaggtagcttttgtgaaataaattcattaatcacttttgtatcatctgttagacgactcattaattggccactttcatttttatcaaagaacggcatttttaattga peg.2217# hypothetical SEQ ID NO: 53 proteinatgattctatggaagaaatatgggagctatgaaatgcaaattgcatttaaagatttcaatgaagataagcaaactattaatgagtatactcattttttagttcagaaagaactacattga peg.2226#hypothetical SEQ ID NO: 55 proteinatggatgattataactctaataatgatacgaatgattggcatgaaatcattgaacagctgaagaacgataacgagatactcaaatctaacaatcaagaacttcagcaacatattcatcagctggaagacgagatagacccaatgagacaagaaaatgatgtttttcatcatttattacaacattttgatagtacagcatttatgaacttcaatacatatcgtgatgatcgcccattaaaaaatgcgattaaacgattgaaagaacaataa

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method for inhibiting the growth, migration, proliferation and/ormetastasis of a pre-cancer, cancer or neoplastic cell or inhibiting apathogen comprising contacting the cell or pathogen with an inhibitingeffective amount of a composition comprising a compound Formula I(a):

or a pharmaceutically acceptable salt or prodrug thereof, wherein, N¹-N⁵are nitrogen atoms; X¹-X² are carbon atoms; the R groups attached by adashed line are present, or are not present if the R group is connectedto an atom that is bound to another atom by a double covalent bond; thebond indicated by both a straight line and a dashed line indicate thatthe bond may be a single covalent bond or a double covalent bond; thefused heterocyclic ring system comprises three double bonds with N² orN³ forming a double bond and with X¹, and with N⁴ or N⁵ forming a doublebond with X²⁻; R¹ is a hydroxyl, ester, carboxylic acid, or —O—R¹⁰, R²,R⁴, R⁵, R⁷-R⁹ are independently a H, D, optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-alkenyl, optionallysubstituted (C₁-C₆)-alkynyl, optionally substituted (C₃-C₁₂)cycloalkyl,optionally substituted (C₄-C₁₂)cycloalkenyl, optionally substitutedaryl; R³ and R⁶ are independently selected from a H, D, optionallysubstituted (C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-heteroalkyl,optionally substituted (C₁-C₆)-alkenyl, optionally substituted(C₁-C₆)-heteroalkenyl, optionally substituted (C₁-C₆)-alkynyl,optionally substituted (C₁-C₆)-heteroalkynyl, optionally substituted(C₃-C₁₂)cycloalkyl, optionally substituted (C₄-C₁₂)cycloalkenyl,optionally substituted aryl, optionally substituted heterocycle, halide,hydroxyl, carbonyl, aldehyde, carboxyl, ester, alkoxy, carboxyamide,amine, imine, azide, cyano, nitro, nitroso, thiol, sulfide, sulfoxide,sulfone, and phosphate; R¹⁰ is selected from D, optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-heteroalkyl, optionallysubstituted (C₁-C₆)-alkenyl, optionally substituted(C₁-C₆)-heteroalkenyl, optionally substituted (C₁-C₆)-alkynyl,optionally substituted (C₁-C₆)-heteroalkynyl, optionally substituted(C₃-C₁₂)cycloalkyl, optionally substituted (C₄-C₁₂)cycloalkenyl,optionally substituted aryl, and optionally substituted heterocycle.2-3. (canceled)
 4. The method of claim 1, wherein the pre-cancer, canceror neoplastic cell is selected from the group consisting of melanoma,squamous cell carcinoma, actinic keratosis, keratoacanthoma, and basalcell carcinoma.
 5. The method of claim 1, wherein the cancer cell iscontacted in vivo.
 6. The method of claim 1, wherein the contacting isthrough topical administration.
 7. The method of claim 1, wherein thecomposition further comprises a chemotherapeutic agent.
 8. The method ofclaim 1, wherein the composition is formulated for topicaladministration.
 9. The method of claim 1, wherein the composition isformulated for systemic administration.
 10. The method of claim 1,wherein the composition comprises a commensal probiotic bacteria thatproduces the compound of Formula I(a), I(b) or II.
 11. The method ofclaim 10, wherein the commensal probiotic bacteria expresses 1 or moreof the sequences set forth in SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,and/or
 55. 12. The method of claim 10, wherein the commensal probioticbacteria comprises S. epidermidis strain MO34 and/or MO38.
 13. A topicalprobiotic composition for producing or maintaining skin microbiomebalance and/or treating infection or neoplasms, the compositioncomprising a therapeutically effective amount or inhibiting effectiveamount of one or more compounds having the structure of Formula I(a):

or a pharmaceutically acceptable salt or prodrug thereof, wherein, N¹-N⁵are nitrogen atoms; X¹-X² are carbon atoms; the R groups attached by adashed line are present, or are not present if the R group is connectedto an atom that is bound to another atom by a double covalent bond; thebond indicated by both a straight line and a dashed line indicate thatthe bond may be a single covalent bond or a double covalent bond; thefused heterocyclic ring system comprises three double bonds with N² orN³ forming a double bond and with X¹, and with N⁴ or N⁵ forming a doublebond with X²; R¹ is a hydroxyl, ester, carboxylic acid, or —O—R¹⁰, R²,R⁴, R⁵, R⁷-R⁹ are independently a H, D, optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-alkenyl, optionallysubstituted (C₁-C₆)-alkynyl, optionally substituted (C₃-C₁₂)cycloalkyl,optionally substituted (C₄-C₁₂)cycloalkenyl, optionally substitutedaryl; R³ and R⁶ are independently selected from a H, D, optionallysubstituted (C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-heteroalkyl,optionally substituted (C₁-C₆)-alkenyl, optionally substituted(C₁-C₆)-heteroalkenyl, optionally substituted (C₁-C₆)-alkynyl,optionally substituted (C₁-C₆)-heteroalkynyl, optionally substituted(C₃-C₁₂)cycloalkyl, optionally substituted (C₄-C₁₂)cycloalkenyl,optionally substituted aryl, optionally substituted heterocycle, halide,hydroxyl, carbonyl, aldehyde, carboxyl, ester, alkoxy, carboxyamide,amine, imine, azide, cyano, nitro, nitroso, thiol, sulfide, sulfoxide,sulfone, and phosphate; R₁₀ is selected from D, optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₁-C₆)-heteroalkyl, optionallysubstituted (C₁-C₆)-alkenyl, optionally substituted(C₁-C₆)-heteroalkenyl, optionally substituted (C₁-C₆)-alkynyl,optionally substituted (C₁-C₆)-heteroalkynyl, optionally substituted(C₃-C₁₂)cycloalkyl, optionally substituted (C₄-C₁₂)cycloalkenyl,optionally substituted aryl, and optionally substituted heterocycle.14-15. (canceled)
 16. The topical probiotic composition of any of claim13, comprising at least one probiotic commensal skin bacteria.
 17. Thetopical probiotic composition of claim 16, wherein the probioticcommensal skin bacteria is S. epidermidis strain MO34 and/or MO38. 18.The topical probiotic composition of claim 13, wherein the compositionis formulated as a lotion, shake lotion, cream, ointment, gel, foam,powder, solid, paste or tincture.
 19. A bandage or dressing comprising atopical probiotic composition of claim
 13. 20. A method of treating skindamage due to UV radiation, comprising contacting the skin with atopical probioic composition of claim
 13. 21. A pharmaceuticalcomposition comprising a compound of Formula I(a), I(b) and/or II andone or more pharmaceutically acceptable carriers.
 22. A pharmaceuticalcomposition of claim 21, further comprising one or more chemotherapeuticagents.
 23. The pharmaceutical composition of claim 21, wherein thecomposition is suitable for oral, parenteral, or topical administration.24. The pharmaceutical composition of claim 23, wherein the topicaldosage form is either in the form of a cream, ointment, gel, spray orlotion.